Role of Hepcidin Hormone in Patients of ß-Thalassemia Major

CONTENTS

INTRODUCTION

AIM OF THE WORK

LITERATURE REVIEW

THALASSEMIA

IRON METABOLISM

HEPCIDIN

SUBJECTS AND METHODS

RESULTS

DISCUSSION

CONCLUSIONS

RECOMMENDATION

SUMMARY

REFERENCES

ACKNOWLEDGMENT

First and foremost, thanks to Allah who helped me to finish this work

I am honered to express my deepest appreciation and profound gratitude to Prof. Dr.Nagelaa Ali Khalifa Professor of clinical & chemical pathology, Faculty of Medicine, Zagazig University, for her kind supervision, encouragement and constant guidance

My deepest thanks and gratefulness to Prof. Dr. Mervat Abdallah Hesham, Professor of Pediatrics, Faculty of Medicine, Zagazig University, for her continuous support and advice

I would like to express my deepest sense of gratitude and obligations to Prof. Dr. Ibtessam Ibrahim Ahmed, Assist.Professor at department of Clinical and Chemical Pathology, Faculty of Medicine, Zagazig University

Lastly, I would like to express my deepest thanks to all my colleges in clinical & chemical pathology department for their help and encouragement

Dr. Abeer Badawy

LIST OF ABBREVIATIONS

BMP: Bone morphogenetic protein

BMT: Bone marrow transplantation

Bp: Base pair

Dcytb: Duodenal cytochrome b

DFO: Desferrioxamine

DMT1: Divalent Metal ion Transporter

FEP: Free Erythrocyte Porphyrin

GVHD: Chronic graft-Versus-Host Disease

HFE: High iron Fe

HIF: Hypoxia-inducible factor

HIV: Human Immune deficiency virus

HJV: Hemojuvelin

HLA: Human leukocyte antigen

LCR: Locus control region

MOD: Multi - organ dysfunctions

NGAL: Neutrophil gelatinase-associated lipocalin

NMD: Nonsense- mediated mRNA decay

Nrmap2: Natural resistance macrophage-associated protein

NTBI: Non-transferrin bound iron

PASP: Pulmonary artery systolic pressure

PHT: Pulmonary hypertension

sTfR: Soluble Transferrin receptors

TfR: Transferrin receptors

UGTIA: Uridine diphosphate-glucoronyl transferase IA

VSS: Volume of distribution at steady state

LIST OF TABLES

Table 1 Main characteristics of genetic iron overload disorders (Deugnier et al., 2008)

Table 2 The clinical data of the studied groups

Table 3 Liver and kidney functions results of the studied groups

Table 4 Complete blood count results of the studied groups

Table 5 Results of iron study of the studied groups

Table 6 Hemoglobin Electrophoresis data

Table 7 Hepcidin concentration levels

Table 8 Ratio between hepcidin and Serum Ferritin

Table 9 Correlation between hepcidin and other parameters

LIST OF FIGURES

Fig. 1 The β-Globin Gene Cluster on the Short Arm of Chromosome 11 (Nancy & Livieri, 1999)

Fig. 2 The Normal Structure of the β-Globin Gene and the Locations and Types of Mutations Resulting in β- Thalassemia (Nancy & Livieri, 1999)

Fig. 3 Effects of Excess Production of Free α-Globin Chains (Nancy & Livieri, 1999)

Fig. 4 Complications of beta thalassemia

Fig. 5 Peripheral blood film in Cooley anemia

Fig. 6 Essential roles of iron (Taketani, 2005)

Fig. 7 Normal distribution and storage of body iron (Andrews, 1999)

Fig. 8 Major pathways of iron transfer between cells and tissues (Andrews, 2000)

Fig. 9 Iron absorption (Andrews, 2000)

Fig. 10 Hepatic Iron Burden over Time and the Effect of Various Hepatic Iron Concentrations in Patients with Thalassemia Major, Homozygous Hemochromatosis, and Heterozygous Hemochromatosis (Nancy & Livieri, 1999)

Fig. 11 Amino acid sequence and a model of the major form of human hepcidin. The amino and carboxy termini are labeled as N and C, The pattern of disulfide linkages between the 8 cysteines is also shown in the amino acid sequence (Ganz, 2003)

Fig. 12 Physiology of hepcidin-ferroportin interaction (Rivera et al., 2005b)

Fig. 13 Normal iron homeostasis mediated by an iron-sensing feedback loop (Wrighting & Andrews, 2008)

Fig. 14 Hepcidin mRNA expression (Nicolas et al., 2002)

Fig. 15 Concentration of hepcidine of patients (in circles) and concentration of hecidin of control (in squares)

Fig. 16 The variations of RBCs, HB, MCV, HCT (PCV) for 40 patients (30 patients and 10 controls)

Fig. 17 Hepcidin and Serum Ferritin for 40 patients (30 patients and 10 controls)

Fig. 18 Correlation between hepcidin and Hb

Fig. 19 Correlation between hepcidin and HCT

Fig. 20 Correlation between hepcidin and MCV

Fig. 21 Correlation between hepcidin and Serium Ferritin

INTRODUCTION

The thalassemias are a heterogeneous group of genetic disorders of haemoglobin synthesis, occurring more frequently in the Mediterranean region, the Indian subcontinent, Southeast Asia, and West Africa .The thalassemias are divided according to their severity into major which is severe and transfusion dependent, intermediate and minor forms of illness. The β-thalassemias are the most important types of thalassemia because they are so common and usually produce severe anemia in their homozygous and compound heterozygous states (Hillmanet al., 2005).

In β-thalassemia major, the neonate is well at birth but develops severe anemia, bone abnormalities, failure to thrive, and life-threatening complications. In many cases, the first signs are pallor, yellow skin and scleras in infants ages 3 to 6 months. Later clinical features, in addition to severe anemia, include splenomegaly or hepatomegaly, with abdominal enlargement, frequent infections, bleeding tendencies (especially toward epistaxis), and anorexia (Fucharoenet al.,2000).

Transfusional iron overload is the most important complication of β- thalassemia and is a major focus of management, which can be prevented by adequate iron chelation. Extensive iron deposits are associated with cardiac hypertrophy and dilatation, degeneration of myocardial fibers (Aessoposet al., 1995; Duet al., 1997).

Hepcidin is a 25-amino-acid iron peptide hormone. Initially identified in human plasma and urine as an anti-microbial molecule. Hepcidin is the key regulator of systemic iron homeostasis and a pathogenic factor in anemia of inflammation and hereditary hemochromatosis. Hepcidin inhibits iron influx into plasma from duodenal enterocytes that absorb dietary iron, from macrophages that recycle iron from senescent erythrocytes and from hepatocytes that store iron (Parket al.,2001).

Iron-Loading anemias are characterized by ineffective erythropoiesis and increased intestinal iron absorption. Erythrocyte transfusions further exacerbate the iron overload. The development of hepcidin- based diagnostics and therapies for iron-loading anemias may offer more effective approaches to prevent the toxicity associated with iron overload. The most common iron- loading anemias are major forms of β-thalassemia (Papanikolaouet al., 2005).

In the presence of systemic iron overload Patients with thalassemia major in whom iron overload was more severe and anemia was partially relieved by transfusions, had urinary hepcidin concentrations that were higher than in thalassemia intermedia. These findings were interpreted as supporting the dominant erythropoietic effect of exogenous hepcidin could prevent the iron overload in iron-Loading anemias (Lorealet al., 2005).

Adamsky and his co-authors (2004) have found that iron overload is less dominant than anaemia in regulating hepcidin expression in the setting of the β-thalassemia major mouse model. The decreased expression of hepcidin may explain the increased absorption of iron in thalassemia.

AIM OF THE WORK

The aim of the present work is to measure hepcidin concentration in patients of β thalassemia major to explain its role in iron metabolism for those patients who have iron overload.

LITERATURE REVIEW

THALASSEMIA

Definition

The thalassemias are a heterogeneous group of genetic disorders of haemoglobin synthesis, all of which result from a reduced rate of production of one or more of the globin chains of haemoglobin. The thalassemias are among the most common genetic disorders worldwide, occurring more frequently in the Mediterranean region, the Indian subcontinent, Southeast Asia, and West Africa (Weatherall & Clegg, 2001). Brief historical review

Thalassemia was first defined in 1925 when Dr-Thomas B.Cooley described five young children with severe anemia, splenomegally, and unusual bone abnormalities and called the disorder erythroblastic or Mediterranean anemia because of circulating nucleated red blood cells and because all of his patients were of Italian or Greek ethnicity (Hillmanet al., 2005).

In Europe, Riette 1925 described Italian children with unexplained mild hypochromic and microcytic anemia in the same year Cooley reported the severe form of anemia later named after him. In addition, Wintrobe and coworkers in the United States reported a mild anemia in both parents of a child with Cooley anemia. This anemia was similar to the one that Riette described in Italy. Only then was Cooley's severe anemia recognized as the homozygous form of the mild hypochromic and microcytic anemia that Riette and Wintrobe described. This severe form was then labeled as thalassemia major and the mild form as thalassemia minor. The word thalassemia is a Greek term derived from thalassa, which means "the sea" (referring to the Mediterranean), and emia, which means "related to blood" (Yaish, 2007).

Classification of the Thalassemias:

First, a clinical classification, which describes the degree of severity. Second, the thalssemia can be defined by the particular globin chain that is synthesized at a reduced rate. Finally, it is now often possible to subclassify them according to defect in the globin chain synthesis (Cohenet al.,2004).

I. Clinical classification of the Thalassemias

The thalassemias are divided according to their severity into major,intermediate and minor forms of illness, thalassemia major which is severe and transfusion dependent , and the symptomless minor forms, which usually represent the carrier state, or trait. Thalassemia intermedia describes conditions that associated with a more severe degree of anemia and splenomegaly than the trait but, not as severe as the major forms to require regular transfusions (Weatherall & Clegg, 2001).

(a) Thalassemia major

Thalassemia major, the severe form of Thalassemia occurs when a child inherits two mutated genes, one from each parent. Children born with Thalassemia major usually develop the symptoms of severe anemia within the first year of life. They lack the ability to produce normal adult hemoglobin (HbA, α2β2). Children with Thalassemia major are so chronically fatigued they fail to thrive and do not grow normally. Left untreated, this disorder will cause bone deformities and eventually will lead to death within the first decade of the child’s life (Talwar & Srivastava, 2004).

(b) Thalassemia Intermedia

Thalassemia Intermedia is a mild form of thalassemia that is caused by the one of the more severe thalassemic genes and one of the milder thalassemic genes. Children with Thalassemia intermedia start to develop symptoms later in life than those with Thalassemia major. They are moderately anemic but a large number of the patients survive without regular blood transfusions. The severity of Thalassemia intermedia isn't determined by hemoglobin levels alone; it also depends on how the individual's feelings,their growth rate and development (Eldor & Rachmilewitz, 2002).

(c) Thalassemia minor

Thalassemia minor, people with a Thalassemia trait in one gene are known as carriers or are said to have Thalassemia minor. The only way to know if they carry the Thalassemia trait is to have a special blood hemoglobin electrophoresis which can identify the gene. The carriers of Thalassemia minor show mild hypochromic microcytic anemia (Talwar & Srivastava, 2004).

Normal Human Hemoglobin:

Function

Hemoglobin main function is carrying oxygen through the body to all of the organs (Morenoet al.,2004).

Hemoglobin synthesis and structure

Hemoglobin synthesis requires the coordinated production of heme and globin. Heme is the prosthetic group that mediates reversible binding of oxygen by hemoglobin. Globin is the protein that surrounds and protects the heme molecule (Handinet al.,2003).

The combination of two alpha chains and two gamma chains form "fetal" hemoglobin (α2γ2 ), termed "hemoglobin F". With the exception of the first 10 to 12 weeks after conception, fetal hemoglobin is the primary hemoglobin in the developing fetus. The combination of two alpha chains and two beta chains form "adult" hemoglobin (α2β2), also called "hemoglobin A". Although hemoglobin A is called "adult", it becomes the predominate hemoglobin within about 18 to 24 weeks of birth (Handinet al.,2003).

The genes that encode the alpha globin chains are on chromosome 16. Those that encode the non-alpha globin chains are on chromosome 11. The alpha complex is called the "alpha globin locus", while the non-alpha complex is called the "beta globin locus" (Handinet al.,2003).

In the first trimester of intrauterine life, ζ, ε, α, and γ chains attain significant levels and in various combinations form Hb Gower I (ζ2ε2), Hb Gower II (α2ε2), Hb Portland (ζ2γ2), and fetal hemoglobin (HbF) (α2γ2 136- G and α2γ2 136-A) .Whereas Hb Gower and Hb Portland soon disappear, HbF persists and forms the predominant respiratory pigment during intrauterine life. Before birth, gamma-chain production begins to wane so that after the age of 6 months postpartum, only small amounts of HbF (< 2%) can be detected in the blood (Woodet al .,2001).

In early intrauterine life, beta-chain synthesis is maintained at a low level but gradually increases to significant concentrations by the end of the third trimester and continues into neonatal and adult life. The synthesis of delta chains remains at a low level throughout adult life (< 3%). Hence during normal development, the synthesis of the embryonic hemoglobins Gower and Portland is succeeded by the synthesis of HbF, which in turn is replaced by the adult hemoglobins, HbA and HbA2 (Hardison , 2001).

Alpha Globin Locus

Each chromosome 16 has two alpha globin genes that are aligned one after the other on the chromosome. For practical purposes, the two alph globin genes (termed α1 and α2) are identical. Since each cell has two chromosomes 16, a total of four alpha globin genes exist in each cell. Each of the four genes produces about one-quarter of the alpha globin chains needed for hemoglobin synthesis. The mechanism of this coordination is unknown. Promoter elements exist 5' to each alpha globin gene. In addition, a powerful enhancer region called the locus control region (LCR) is required for optimal gene expression. (Hoff brand, 2006).

Beta Globin Locus

The genes in the beta globin locus are arranged sequentially from 5' to 3' beginning with the gene expressed in embryonic development (the first 12 weeks after conception; called episolon). The beta globin locus ends with the adult beta globin gene. The sequence of the genes is: epsilon, gamma, delta, and beta. There are two copies of the gamma gene on each chromosome 11. The others are present in single copies. Therefore, each cell has two beta globin genes, one on each of the two chromosomes 11 in the cell. These two beta globin genes express their globin protein in a quantity that precisely matches that of the four alpha globin genes. The mechanism of this balanced expression is unknown (Hoffbrand, 2006).

Upstream of the entire β globin complex is the locus control region (LCR), which is essential for the expression of all the genes in the complex (Fig. 1). The general structure of the β globin gene is typical of the other globin loci. The genomic sequence spans 1600 bp and codes for 146 amino acids; the transcribed region is contained in three exons separated by two introns or intervening sequences.The first exon encodes amino acid 1 to 29 together with the first two bases for codon 30, exon 2 encodes part of residue 30 together with amino acids 31 to 104, and exon 3, amino acids 105 to 146 (Hardison, 2001).

Exon 2 encodes the residues involved in heme binding and αβ dimer formation, while exons 1 and 3 encode for the non- heme- binding regions of the β globin chain.The β globin gene promoter includes 3 positive cis- acting elements: TATA box, CCAAT box and duplicated CACCC motifs. In addition to these motifs, the region upstream of the β globin promoter contains two binding motifs for the erythroid transcription factor GATA 1(Mariniet al., 2004).

illustration not visible in this excerpt

Fig. 1 The β-Globin Gene Cluster on the Short Arm of Chromosome 11 (Nancy & Livieri, 1999).

In Panel A, the β-globin-like genes are arranged in the order during development. Panel B shows the timing of the normal developmental switching of human hemoglobin.

Conserved sequences important for gene function are found in the 5’promoter region, at the exon-intron junction, and in the 3 untranslated regions (3-UTR) at the end of the mRNA sequences. The 5’ untranslated region (UTR) occupies a region of 50 nucleotides between the CAP site, the start of transcription, and the initiation (ATG) codon. There are two prominently conserved sequences in the 5’ UTR of the various globin genes (both α and β). The 3’ UTR constitutes the region between the termination codon (TAA) and the poly (A) tail. It consists of 132 nucleotides with one conserved sequence, AATAAA, located 20 nucleotides upstream of the poly (A) tail (Forget et al., 2001).

II. Genetic Classification of the Thalassemias

a) Alpha-thalassemia

Alpha thalassemia syndromes can be expressed as α0 and α+. In the α0, no alpha chains are produced. In the α+, the output of one of the linked pair of alpha-globin genes is defective, and only some alpha chains are produced. Within these general categories of the alpha-thalassemia syndromes, there is considerable genetic and clinical heterogeneity due to the interaction of the many possible mutations directing globin chain synthesis (Rimoinet al.,2006).

Silent Carrier (α+-thalassemia carriers):

The loss of one gene diminishes the production of the alpha protein slightly. This condition is so close to normal that it can be detected only by specialized laboratory techniques which, until recently, were confined to research laboratories. A person with this condition is called a "silent carrier" because of the difficulty of detection (Sabella & Cunningham, 2006).

α0-Thalassemia minor or trait:

The loss of two genes produces a condition with small red blood cells, and at most a mild hypochromic anemia. People who have this condition look and feel normal, but thecondition can’t be detected certainly except by DNA analysis (Sabella & Cunningham, 2006).

Hemoglobin H disease:

Deficiency of 3 α chains leads to production of excess β chains, forms β4-tetramer and produces a serious hematological problem. Patients with this condition have a severe anemia, and often require blood transfusions to survive (Sabella & Cunningham, 2006).

Hemoglobin Bart’s hydrops syndrome:

The loss of all four alpha genes during intrauterine life results in γ4- tetramers, produces a condition that is incompatible with life. Foetus with four-gene deletion alpha thalassemia die in utero or shortly after birth. Rarely, four-gene deletion alpha thalassemia has been detected in utero, usually in a family where the disorder occurred in an earlier child. In utero blood transfusions have saved some of these children. These patients require life-long transfusions and other medical support (Sabella & Cunningham, 2006).

b) Delta-Thalassemia

A hereditary disorder characterized by reduced or absent delta-globin thus effecting the level of hemoglobin A2 (Bouvaet al., 2006).

c) Delta-beta-thalassemia

In δβ+ thalassemia, mutational basis is due to extensive deletions of delta and beta globin structural genes. Abnormal hemoglobin (Hb Lepore) is produced due to unequal crossing over between mispaired δ and β globin genes leading to δ and β fusion with segments of δ, β lost (Weatheral & Clegg, 2000).

d) Beta-thalassemia

Definition:

The β-thalassemias are the most important types of thalassemia because they are so common and usually produce severe anemia in their homozygous and compound heterozygous states. The fact that there are only two genes for the beta chain of hemoglobin makes beta thalassemia a bit simpler to understand than alpha thalassemia. (Hillmanet al., 2005).

Local Prevalence and geographic distribution of β -thalassemia (in Egypt)

El-Beshlawy stated that β-thalassemia is the most common chronic hemolytic anemia in Egypt (85.1%). A carrier rate of 9-10.2% has been estimated in 1000 normal random subjects from different geographical areas of Egypt (El-Beshlawy, 1999).

International Prevalence and geographic distribution of β -thalassemia

Worldwide, 15 million people have clinically apparent thalassemic disorders. People who carry thalassemia in India alone number approximately 30 million. These facts confirm that thalassemias are among the most common genetic disorders in humans. β thalassemia is much more common in Mediterranean countries such as Greece, Italy, and Spain. Many Mediterranean islands, including Cyprus, Sardinia, and Malta, have a significantly high incidence of severe β thalassemia, constituting a major public health problem. For instance, in Cyprus, 1 in 7 individuals carries the gene, which translates into 1 in 49 marriages between carriers and 1 in 158 newborns expected to have β thalassemia major. As a result, preventive measures established and enforced by public health authorities have been very effective in decreasing the incidence among their populations. β thalassemia is also common in North Africa, the Middle East, India, and Eastern Europe. Conversely, β thalassemia is more common in Southeast Asia, India, the Middle East, and Africa (Yaish, 2007).

Classification of β- thalassemia:

It occurs in three clinical forms: major, intermediate, and minor. The resulting anemia’s severity depends on whether the patient is homozygous or heterozygous for the thalassemic trait (Rund & Rachmilewitz, 2005).

Molecular basis of β thalassemia

There are two main varieties of β thalassemia alleles; β 0 thalassemia in which no β globin is produced, and β+ thalassemia in which some β globin is produced, but less than normal. In contrast to the α thalassemias, the β thalassemias are rarely caused by deletions. One group of deletions affects only the β globin gene and ranges in size from 290 bp to > 60 Kb. Of these, only the 619 bp deletion at the 3’ end of the β gene is common (Thein, 1998).

The other deletions, although extremely rare, are of particular functional and phenotypic interest because they are associated with unusually high levels of Hb A2 in heterozygotes. These deletions differ widely in size, but remove in common a region from positions -125 to +78 relative to the mRNA cap site in the β promoter which includes the CACCC, CCAAT and TATA elements (Forgetet al.,2001).

The mechanism underlying the markedly elevated levels of Hb A2 appears to be related to the removal of the 5’ promoter region of the β gene. This may remove competition for the upstream LCR leading to its increased interaction with the γ and δ genes in cis, enhancing their expression (Wood et al .,2001).

There is a disproportionate increase of Hb A2 (α2 δ2) derived from the δ globin gene cis to the β globin gene deletion. This mechanism may also explain the moderate increases in Hb F which characterize this group of deletions and those due to point mutations affecting the promoter region. Although the increases in Hb F are variable, and modest in heterozygotes, they are adequate to compensate for the complete absence of β globin in homozygotes. Two homozygotes for different deletions of this kind have a mild disease despite the complete absence of Hb A2 (α2 β2) (Craiget al ., 1992).

The vast majority of β thalassemias are caused by point mutations within the gene or its immediate flanking sequences (Fig. 2). These single base substitutions, minor insertions or deletions of a few bases are classified according to the mechanism by which they affect gene regulation: transcription, RNA processing or RNA translation. Mutations affecting transcription can involve either the conserved DNA sequences that form the β globin promoter or the stretch of 50 nucleotides in the 5’UTR. Generally they result in a mild to minimal deficit of β globin output and can be silent in carriers (Theinet al., 2001).

illustration not visible in this excerpt

Fig. 2 The Normal Structure of the β-Globin Gene and the Locations and Types of Mutations Resulting in β-Thalassemia (Nancy & Livieri, 1999).

All β-globin-like genes contain three exons and two introns between codons 30 and 31 and 104 and 105, respectively. Approximately half of the β thalassemia alleles affect the different stages of RNA translation and in all instances, no β globin is produced resulting in β0 thalassemia. Most of these defects result from the introduction of premature termination codons due to frameshifts or nonsense mutations and nearly all terminate within exon 1 and 2. Mutations that result in premature termination early in the sequence (in exons 1 and 2) are associated with minimal steady-state levels of β mRNA in erythroid cells, due to an accelerated decay of the abnormal mRNA referred to as nonsense- mediated mRNA decay (NMD) (Maquat,1995) .

Variants of β thalassemia

Dominantly inherited β thalassemia

In contrast to the common β thalassemia alleles that are prevalent in malarial regions and inherited typically as Mendelian recessives, some forms of β thalassemia are dominantly inherited, in that inheritance of a single β thalassemia allele results in a clinically detectable disease despite a normal α globin genotype. Heterozygotes have a thalassemia intermedia phenotype with moderate anemia, splenomegaly and a thalassemic blood picture. Apart from the usual features of heterozygous β thalassemia, such as increased levels of HbA2 and the imbalanced α/β globin biosynthesis, large inclusion bodies similar to those seen in thalassemia major are often observed in the red cell precursors, hence the original term of inclusion body thalassemia (Feiet al .,1989).

Normal Hb A2 β thalassemias

The diagnostic feature of β thalassemia is the hypochromic microcytic red cells and an elevated level of Hb A2 in heterozygotes, whether β+ or β0. Normal Hb A2 β thalassemias refer to the forms in which the blood picture is typical of heterozygous β thalassemia except for the normal levels of Hb A2. Most cases result from co-inheritance of δ thalassemia (δ0 or δ+) in cis or trans to the β thalassemia gene, which can be of the β0 or β+ type. One relatively common form of normal Hb A2 thalassemia is that associated with Hb Knossos. The mutation activates an alternative splice site reducing the amount of normal transcript that contains the variant (Trifilliset al ., 1991).

Another fairly common cause of normal Hb A2 β thalassemia phenotype in the Greek population is the Corfu form of δ β thalassemia. The phenotype of normal Hb A2 β thalassemia is also seen in heterozygotes for ε γ δ β thalassemia and overlaps the phenotypes encountered in carriers of α thalassemia (Trifilliset al .,1991).

Silent β thalassaemia

The silent β thalassemias cause only a minimal deficit of β globin production. Heterozygotes do not have any evident hematologic phenotype; the only abnormality being a mild imbalance of globin chain synthesis. These mutations have been identified in homozygotes who have a typical β thalassemia trait phenotype or in the compound heterozygous state with a severe β thalassemia allele where they cause thalassemia intermedia (Gonzalez-Redondoet al .,1989).

β thalassemia trait with unusually high Hb A2

Despite the vast heterogeneity of mutations, the increased levels of Hb A2 observed in heterozygotes for the different β thalassemia alleles in different ethnic groups are remarkably uniform, usually 3.5-5.5% and rarely exceeding 6%. Unusually high levels of Hb A2 over 6.5% seem to characterize the sub-group of β thalassemias caused by deletions that remove the regulatory elements in the β promoter (Codringtonet al ., 1990).

β thalassemia due to insertion of a transposable element

Transposable elements may occasionally disrupt human genes and result in their inactivation. The insertion of such an element, a retrotransposon of the family called L1 has been reported with the phenotype of β + thalassemia (Divokyet al .,1996).

β thalassemia due to trans-acting determinants

Population studies have shown that ~1% of the β thalassemias remain uncharacterized despite extensive sequence analysis, including the flanking regions of the β globin genes. In several families, linkage studies demonstrated that the β thalassemia phenotype aggregates independently of the β globin complex implying that the genetic determinant is trans-acting (Giordanoet al ., 1998).

Somatic deletion of β globin gene

This novel mechanism was recently described in an individual who had moderately severe thalassemia intermedia despite being constitutionally heterozygous for β0 thalassemia with a normal genotype. Subsequent investigations revealed that he had a somatic deletion of a region of chromosome 11p15 including the β globin complex giving rise to a mosaic of cells, 50% with one and 50% without any β globin gene. The sum total of the β globin product is ~25% less than the normally asymptomatic β thalassemia trait (Badenset al .,2002).

Clinical features of severe β-thalassemic syndromes

Infants and children affected with β thalassemia have pallor, poor development, and abdominal enlargement. Hemoglobin electrophoretic patterns show a variable quantity of HbA2 (0% - 6%) depending on the genotype of the patient. The anemia is due to a combination of ineffective erythropoiesis, excessive peripheral red blood cell hemolysis, and progressive splenomegaly (Weatherall & Clegg, 2000). The latter causes an increase in plasma volume and a decrease in total red cell mass. The red cells are microcytic (mean corpuscular volume <70 fL) with marked anisochromasia. The bone marrow shows marked erythroid hyperplasia, and the serum ferritin level is elevated (Wonke, 2001).

In children and young adults, radiologic abnormalities include thinning of the long bones with sun-ray appearance and dilatation of the marrow cavities. The skull has a “hair-on-end” appearance because of widening in the diploic space. Patients with thalassemia have enlarged maxillary sinuses and tend to have a maxillary overbite. The face gradually assumes a “mongoloid” appearance. Such changes promote infections in the ears, nose, and throat. Because of chronic anemia and iron overload, endocrinopathies such as hypopituitarism, hypothyroidism, hypoparathyroidism, diabetes mellitus, cardiomyopathy, and testicular or ovarian failure become common as the child with thalassemia grows older (Cunninghamet al., 2004; Rund & Rachmilewitz, 2005).

Thalassemia can be regarded as a chronic hypercoagulable state. Venous and arterial thromboembolic phenomena tend to occur more frequently in thalassemic patients who have undergone splenectomy. Furthermore, such patients may develop progressive pulmonary arterial disease due to platelet thrombi in the pulmonary circulation (Eldor & Rachmilewitz, 2002).

Pathophysiology

Mechanisms of Anemia

Normal hemoglobin, hemoglobin A, is composed of 2 beta and 2 alpha subunits. In beta thalassemia major, more than 200 mutations have been described in the beta-globin genes, cause loss of both beta-globin subunits.

This leaves the normally paired alpha subunits unpaired. Unpaired subunits are catatonic (Handinet al., 2003).

Normally, compensatory mechanisms are present to protect the cell from the small amounts of unpaired alpha subunits, which may regularly be present. This significant excess of free α chains caused by the deficiency of β chains causes destruction of the RBC precursors in the bone marrow (i.e., ineffective erythropoiesis) (Fig. 3). This ineffective erythropoiesis and profound hemolysis result in a severe anemia that is usually manifest in affected individuals by age 6 months (Rimoinet al., 2006).

illustration not visible in this excerpt

Fig. 3 Effects of Excess Production of Free α-Globin Chains (Nancy & Livieri, 1999).

The physiologic response is to attempt to increase red cell production by expanding the bone marrow space up to 30-fold and/or increase production of non-beta hemoglobin chains such as A2 (δ) and fetal (γ) hemoglobin. However, despite these mechanisms, erythropoiesis remains ineffective and these patients become transfusion-dependent early in life. In fact, the presence or absence of adequate transfusions significantly impacts the appearance of these patients and the course of the disease (Sabella & Cunningham, 2006).

Excess unbound α-globin chains and their degradation products precipitate in red-cell precursors, causing defective maturation and ineffective erythropoiesis. Hemolysis Anemia stimulates the synthesis of erythropoietin, leading to an intense proliferation of the ineffective marrow, which in turn causes skeletal deformities and a variety of growth and metabolic abnormalities (Cunninghamet al., 2004).

Clinical features of β-thalassemia major

In β-thalassemia major, the neonate is well at birth but develops severe anemia, bone abnormalities, failure to thrive, and life-threatening complications. In many cases, the first signs are pallor and yellow skin and scleras in infants ages 3 to 6 months. Later clinical features, in addition to severe anemia, include splenomegaly or hepatomegaly, with abdominal enlargement, frequent infections, bleeding tendencies (especially toward epistaxis), and anorexia (Fucharoenet al.,2000).

Children with thalassemia major typically have small bodies and large heads and may also be mentally retarded. Infants may have mongoloid features because bone marrow hyperactivity has thickened the bone at the base of the nose. As these children grow older, they become susceptible to pathologic fractures as a result of expansion of the marrow cavities with thinning of the long bones. They’re also subject to cardiac arrhythmias, heart failure, and other complications that result from iron deposits in the heart and in other tissues from repeated blood transfusions (Fucharoenetal.,2000).

Complications of β-thalassemia major

Iron overload of tissue is the most important complication of β- thalassemia and is a major focus of management. Thalassemia major can be complicated with CCF, hepatic failure, aplastic crisis, intercurrent infection, growth retardation, delayed puberty, hemosiderosis and hemochromatosis. Transfusion related infection (HIV, HB, HC), complications related to ironchelation therapy, endocrinopathies (diabetes mellitus, hypothyroidism, hypogonadism), skeletal complications and multiorgan dysfunctions (MOD) also may found (Fig. 4) (Deugnieret al.,2008).

illustration not visible in this excerpt

Fig. 4 Complications of beta thalassemia.

Management of thalassemia and treatment-related complications (Cunninghamet al., 2004).

Hyperbilirubinemia and a propensity to gallstone formation is a common complication of β thalassemia and is attributed to the rapid turnover of the red blood cells, bilirubin being a break-down product of hemoglobin. Studies have shown that the levels of bilirubin and the incidence of gallstones in β thalassemia, from trait to major, is related to a polymorphic variant (seven TA repeats) in the promoter of the uridine diphosphate-glucoronyltransferase IA (UGTIA) gene, also referred to as Gilbert’s syndrome (Sampietroet al.,1997).

Cardiac iron overload is the most frequent cause of death from chronic transfusion therapy. Recurrent pericarditis may be the initial manifestation of myocardial iron deposition. Ventricular tachycardia and fibrillation or severe congestive heart failure often proves fatal . Cardiac complications, such as pulmonary hypertension (PHT), are the leading cause of death in beta-thalassemia patients. L-Carnitine, due to its role in fatty acid oxidation, might help control the elevation in pulmonary artery systolic pressure (PASP) (El-Beshlawyet al.,2008).

Causes of death in patients with Beta - thalassemia major:

The prognosis of patients with homozygous β-thalassemia major has been improved by transfusion and iron-chelation therapy. Prognosis for survival without cardiac disease is excellent for patients who receive regular transfusions and whose serum ferritin concentrations remain below 2500ng per milliliter with chelation therapy (Borgna-Pignatti et al., 2004).

The most common cause of death in patients with beta thalassemia is heart failure followed by infection, liver cirrhosis, thrombosis, cancer, and diabetus (Borgna-pignatti et al., 2004).

Laboratory diagnosis of β-thalassemia major

The CBC count and peripheral blood film examination results are usually sufficient to suspect the diagnosis. Hb evaluation confirms the diagnosis in β thalassemia, Hb H disease, and Hb E/β thalassemia.

- In the severe forms of thalassemia, the Hb level ranges from 2-8 g/dL.
- MCV and MCH are significantly low, but, unlike thalassemia trait, thalassemia major is associated with a markedly elevated RDW, reflecting the extreme anisocytosis.
- The WBC count is usually elevated in β thalassemia major; this is due, in part, to miscounting the many nucleated RBCs as leukocytes. Leukocytosis is usually present, even after excluding the nucleated RBCs. A shift to the left is also encountered, reflecting the hemolytic process.
- Elevated reticulocytic count.
- Platelets count is usually normal, unless the spleen is markedly enlarged.
- Peripheral blood film examination reveals marked hypochromasia and microcytosis, hypochromic macrocytes that represent the polychromatophilic cells, nucleated RBCs, basophilic stippling, and occasional immature leukocytes shown in (Fig. 5)
- In thalassemia major, laboratory results show elevated bilirubin, urinary and fecal urobilinogen levels (Wonke, 2001).
- Hb electrophoresis and alkali denaturation test reveal an elevated Hb F fraction, which is distributed heterogeneously in the RBCs of patients with β thalassemia, Hb H in patients with Hb H disease, and Hb Bart in newborns with β thalassemia trait. In β -0 β, no Hb A is usually present; only Hb A2 and Hb F are found (Wonke, 2001).
- Free erythrocyte porphyrin (FEP) tests may be useful in situations in which the diagnosis of beta thalassemia minor is unclear. FEP level is normal in patients with the beta thalassemia trait, but it is elevated in patients with iron deficiency or lead poisoning (Wonke, 2001).
- Decreased hepcidin level in patients with β thalassemia major.

illustration not visible in this excerpt

Fig. 5 Peripheral blood film in Cooley anemia.

Iron studies are as follow:

Serum iron level is elevated, with saturation reaching as high as 80% with decreased total iron binding capacity. The serum ferritin level, which is frequently used to monitor the status of iron overload, is also elevated. However, an assessment using serum ferritin levels may underestimate the iron concentration in the liver of a transfusion-independent patient with thalassemia. Increased levels of transferrin receptors (TfR) and soluble TfR (sTfR). Complete RBC phenotype, hepatitis screen, folic acid level, and human leukocyte antigen (HLA) typing are recommended before initiation of blood transfusion therapy (Hillmanet al.,2005).

Management of B-Thalassemia Major

1- Regular blood transfusion and chelation therapy:

Regular transfusion therapy to maintain hemoglobin levels of at least 9 to 10 g per deciliter allows for improved growth and development and also reduces hepatosplenomegaly due to extramedullary hematopoiesis as well as bone deformities (Cunninghamet al.,2004 & Oldet al.,2001). Choice of the scheme for blood transfusion in thalassemia major:

(a) Intermediate schemes: mean Hb 9-10 gm/dl are acceptable in terms of daily living.
(b) Hypertransfusion schemes: mean Hb 10 gm/dl or greater, improve the quality of life without accelerating the lethal complication of iron overload
(c) Supertransfusion program: Maintaince of mean hemoglobin level at 11-12 gm/dl Hb level is not allowed to drop below 12 gm/dl and is raised regularly to 14 by transfusion every 2-3 weeks supertransfusion permits an excellent quality of life (Cohenet al., 2004).

Prevention of Secondary Complications

The most common secondary complications are those related to transfusional iron overload, which can be prevented by adequate iron chelation. After ten to 12 transfusions, chelation therapy is initiated with desferrioxamine B (DFO) administered five to seven days a week by 12- hour continuous subcutaneous infusion via a portable pump. Recommended dosage depends on the individual's age and the serum ferritin concentration. Young children start with 20-30 mg/kg/day, increasing up to 40 mg/kg/day after age five to six years. The maximum dose is 50 mg/kg/day after growth is completed. The dose may be reduced if serum ferritin concentration is low. By maintaining the total body iron stores below critical values (i.e., hepatic iron concentration <7.0 mg per gram of dry weight liver tissue), desferrioxamine B therapy prevents the secondary effects of iron overload, resulting in a consistent decrease in morbidity and mortality (Borgna- Pignattiet al.,2004).

The survival of individuals who have been well transfused and treated with appropriate chelation extends beyond age 30 years. Offbrand et al (2003); said that iron-chelation therapy is largely responsible for doubling the life expectancy of patients with thalassemia major but Deferoxamine continues to be the most common iron-chelating agent in use, but it has several limitations: the need for parenteral administration (which is painful and reduces compliance), side effects, and cost (which is prohibitive in underdeveloped countries) (Borgna-Pignattiet al., 2004).

Much effort has been invested in the development of new orally active chelators. Deferiprone, an orally administered chelator, was initially thought to be an inadequate chelator that might worsen hepatic fibrosis. However, cumulative worldwide experience indicates that the drug is safe and effective. Long-term administration of deferiprone does not appear to be associated with liver damage (Wanlesset al., 2003). Adverse effects of deferiprone include arthralgia, nausea and other gastrointestinal symptoms, fluctuating liver enzyme levels, leukopenia, and rarely agranulocytosis and zinc deficiency. Most of these effects can be monitored and controlled (Ceciet al.,2002)..

Overly vigorous chelation is associated with deferoxamine-induced bone dysplasia, which can slow growth velocity in children and may be only partially reversible (Rund & Rachmilewitz, 2005).

Deferasirox recently became available for clinical use in patients with thalassemia. It is effective in adults and children and has a defined safety profile that is clinically manageable with appropriate monitoring. The most common treatment-related adverse events are gastrointestinal disorders, skin rash, and a mild, non-progressive increase in serum creatinine concentration. Post-marketing experience and several phase IV studies will further evaluate the safety and efficacy of deferasirox. New strategies of chelation using a combination of desferrioxamine and deferiprone have been effective in individuals with severe iron overload; toxicity was manageable (Wuet al.,2004; Tanneret al.,2007).

2- Bone marrow transplantation

Bone marrow transplantation (BMT) from an HLA-identical sib represents an alternative to traditional transfusion and chelation therapy. If BMT is successful, iron overload may be reduced by repeated phlebotomy, thus eliminating the need for iron chelation. The outcome of BMT is related to the pretransplantation clinical conditions, specifically the presence of hepatomegaly, extent of liver fibrosis, and magnitude of iron accumulation. In children who lack the above risk factors, disease-free survival is over 90% (Gaziev & Lucarelli, 2003).

A lower survival rate of approximately 60% is reported in individuals with all three risk factors. Chronic graft-versus-host disease (GVHD) of variable severity may occur in 5%-8% of individuals. BMT from unrelated donors has been carried out on a limited number of individuals with β- thalassemia. Provided that selection of the donor is based on stringent criteria of HLA compatibility and that individuals have limited iron overload, results are comparable to those obtained when the donor is a compatible sib. However, because of the limited number of individuals enrolled, further studies are needed to confirm these preliminary findings (La Nasaet al.,2005).

3- Cord blood transplantation.

Cord blood transplantation from a related donor offers a good probability of a successful cure and is associated with a low risk of GVHD (Locatelliet al.,2003; Walterset al.,2005). For couples who have already had a child with thalassemia and who undertake prenatal diagnosis in a subsequent pregnancy, prenatal identification of HLA compatibility between the affected child and an unaffected fetus allows collection of placental blood at delivery and the option of cord blood transplantation to cure the affected child. On the other hand, in case of an affected fetus and a previous normal child, the couple may decide to continue the pregnancy and pursue BMT later, using the normal child as the donor (Orofinoet al., 2003).

4- Hematopoietic Stem-Cell Transplantation

Although hematopoietic stem-cell transplantation is the only available curative approach for thalassemia, it has been limited by the high cost and the scarcity of HLA-matched, related donors. The past several years have brought progress in the realms of conditioning regimens, donor identification and selection, and the development of alternative sources of hematopoietic stem cells (Talwar & Srivastava, 2004).

5- Splenectomy

Splenectomy is usually not needed if regular transfusion therapy is followed. If the child already has a big spleen ( his transfusion requirement increases to more than times of normal or more than 200 ml packed red cells or over 400 ml of whole blood per kg), splenectomy is indicated . Portal vein thrombosis is a recognized complication after splenectomy due to hypercoagulable state in thalassemia (Wonke, 2001).

6- Diet and vitamins:

No strict regulations regarding diet can be recommended. However, food rich in iron e.g., meat, liver, kidney and green leafy vegetables should be avoided.Diet should include food high in phosphorus or phytates e.g., cereals bread, milk, soya beans, roasted peas, etc. to inhibit iron absorption. Similarly tea can be taken along within an hour after meals to reduce iron absorption (Wonke, 2001).

7- Vitamin C

Ascorbate repletion (daily dose not to exceed 100-150 mg) increases the amount of iron removed after DFO administration. Vitamin C facilitates iron chelation with DFO and should be supplemented in patients receiving DFO (5 mg/kg/d maximum of 200 mg/d). However, in unchelated patients a low vitamin C status is beneficial (Wonke, 2001).

8- Folic acid

Folic acid (5 mg per week) should be given to patients receiving on or irregular transfusion. This is because of relative folate deficiency due to increasesd folate consumption. However,patients receiving regular blood transfusions ordinarily do not require folic acid unless actual deficiency state exist (Wonke, 2001).

Other therapies

1- Combination therapy and Induction of fetal hemoglobin synthesis

New chelation strategies, including the combination or alternate treatment with the available chelators, are under investigation. Induction of fetal hemoglobin synthesis can reduce the severity of β-thalassemia by improving the imbalance between alpha and non-alpha globin chains. Several pharmacologic compounds including 5-azacytidine, decytabine, and butyrate derivatives have had encouraging results in clinical trials. These agents induce Hb F by different mechanisms that are not yet well defined. Their potential in the management of β-thalassemia syndromes is under investigation (Pace & Zein, 2006).

2- Hydroxyurea treatment

The efficacy of hydroxyurea treatment in individuals with thalassemia is still unclear. Hydroxyurea is used in persons with thalassemia intermedia to reduce extramedullary masses, to increase hemoglobin levels, and, in some cases, to improve leg ulcers. A good response, correlated with particular polymorphisms in the beta-globin cluster (i.e., C > T at -158 G gamma), has been reported in individuals with transfusion dependence. However, controlled and randomized studies are warranted to establish the role of hydroxyurea in the management of thalassemia syndromes (Bradaiet al., 2003 ).

3- Correction of molecular defects

The possibility of correction of the molecular defect in hematopoietic stem cells by transfer of a normal gene via a suitable vector or by homologous recombination is being actively investigated (Sorrentino & Niehuis, 2001).

Initial efforts at gene therapy were directed against diseases of the β - globin gene. This therapeutic strategy involves the insertion of a normally functioning β -globin into the patient's autologous hematopoietic stem cells (Puthenveetil & Malik 2004).

The major problems with this type of gene therapy have been related to vector construction. The genetic elements of the vector that are necessary for appropriate regulation of the inserted gene have been defined .However, the therapeutic gene must be inserted into a hematopoietic stem cell and must be expressed at high levels, over an extended period, in an erythroid- specific manner. In addition, the vector must be safe from recombination or mutagenesis. Oncoretroviral and adenoviral vectors have been found to be unsuitable for various reasons) (Puthenveetil &Malik 2004).

Recombinant human erythropoietin was shown to provide the benefit of increasing "thalassemic erythropoiesis" without raising fetal hemoglobin. The effect appeared to be dose-dependent and was observed primarily in patients with thalassemia intermedia who had undergone splenectomy (Personset al.,2003).

Recently, long-acting darbepoetin alfa was shown to increase hemoglobin levels substantially in patients with hemoglobin E β- thalassemia disease. Two important obstacles to the use of recombinant human erythropoietin are its relatively high cost and its subcutaneous administration route, which restrict its use in developing countries. Appropriate clinical protocols are needed to delineate the role of recombinant human erythropoietin (alone or in combination with the aforementioned drugs) in the treatment of thalassemia (Personset al., 2003).

IRON METABOLISM

Iron is an essential nutrient that is required for the oxygen-carrying capacity of hemoglobin. Failure to incorporate adequate iron into heme results in impaired erythrocyte maturation, leading to microcytic, hypochromic anemia. Therefore, circulating factors that modulate iron availability are of major importance in erythropoiesis. Normally, the total body iron endowment is maintained within a tight range between 3 and 5 g (Andrews, 2000).

Systemic iron is distributed among erythrocyte precursors in the bone marrow, tissue macrophages, liver, and all other tissues, with the largest amount found in circulating erythrocytes. Homeostasis is maintained by regulating the levels of plasma iron. Hepcidin, a circulating peptide hormone, has recently emerged as a key modulator of plasma iron concentration and thus, a central regulator of iron homeostasis (Nemeth, 2004).

The cross-talk which has taken place in recent years between clinicians and scientists has resulted in a greater understanding of iron metabolism with the discovery of new iron-related genes including the hepcidin gene which plays a critical role in regulating systemic iron homeostasis. Consequently, the distinction between (a) genetic iron-overload disorders including haemochromatosis related to mutations in the HFE, hemojuvelin, transferrin receptor 2 and hepcidin genes and (b) non-haemochromatotic conditions related to mutations in the ferroportin, ceruloplasmin, transferrin and di-metal transporter 1 genes, and (c) acquired iron-overload syndromes has become easier (Lorealet al., 2005).

Iron is one of the most common elements in nature and a transition metal , iron is involved in electron transport and maintenance of the respiratory chain , it is required for the functioning of proteins involved in ( oxidative energy production, oxygen transport, mitochondrial respiration and inactivation of harmful oxygen radicals) , it is essential for the synthesis of hemoglobin and myoglobin , it plays an important role in detoxification of the reactive species and it is rate limiting in DNA synthesis (Fig. 6) (Taketani , 2005).

In a normal balanced state, 1-2 mg of iron enters and leaves the body every day. Dietary iron is absorbed by duodenal enterocytes and circulates in the plasma bound to transferrin, the main iron transport protein. Most of the circulating iron is used by the bone marrow to generate hemoglobin for red blood cells, while around 10-15% is utilized by muscle fibers to generate myoglobin. Iron released by tissue breakdown is absorbed and recycled. Excess iron is stored by parenchymal cells in the liver and reticuloendothelial macrophages. Traces of iron are lost each day by sloughing of mucosal cells, loss of epithelial cells and any blood loss. Since the human body has not evolved a mechanism to clear excess iron, disorders of iron balance, such as iron overload, are among the most common diseases in humans (Andrews, 1999).

illustration not visible in this excerpt

Fig. 6 Essential roles of iron (Taketani, 2005).

In a normal state, once iron has been absorbed it is complexed with transferrin . Because of the important role of iron in metabolism, the human body has many mechanisms to absorb, transfer and store iron, but none to excrete excess iron Although serum ferritin levels are indicative of body iron levels, a number of conditions can alter the correlation between serum ferritin levels and body iron stores. Acute and chronic inflammation and infections can greatly influence levels; ascorbate levels and increased erythropoiesis can also affect circulating ferritin levels (Fig. 7) (Fleming and Bacon, 2005).

illustration not visible in this excerpt

Fig. 7 Normal distribution and storage of body iron (Andrews, 1999).

Iron Metabolism and Homeostasis

Iron metabolism

Nearly all circulating iron is bound by the abundant serum glycoprotein transferrin. Transferrin can carry one (monoferric transferrin) or two (holotransferrin) atoms of iron per protein molecule. Erythroid precursors are the primary consumers of circulating transferrin-bound iron. During differentiation, changing rates of hemoglobin production correlate with variations in the cell surface complement of transferrin receptor-1 (TFR1). Fluctuations in TFR1 expression control the amount of transferrin-bound iron entering into erythroid cells (Chan &Gerhardt, 1992).

The process by which transferrin delivers iron to these cells is called the transferrin cycle. Upon binding to TFR1 at the cell surface, transferrin and its iron are endocytosed. These endosomal compartments are actively acidified by proton pumps. Acidification facilitates iron release from transferrin because low pH decreases the affinity of the protein for iron. Iron then leaves the endosome through divalent metal ion transporter 1 (DMT1, also known as Nramp2 and SLC11A2) to become available for heme biosynthesis (Canonne-Hergauxet al., 2001).

The insertion of iron into protoporphyrin IX, the final step in heme biosynthesis, occurs in the mitochondrion. Mitoferrin, a protein mutated in anemic zebra fish, was recently shown to act as a mitochondrial iron importer necessary for heme biosynthesis . However, another group has postulated that iron is transferred from endosomes directly into mitochondria through direct membrane contacts between the organelles. The fates of TFR1 and apotransferrin are more certain—they are recycled to the cell surface and circulation, respectively, where they repeat the cycle (Sheftel, 2007).

The amount of circulating, transferrin-bound iron is determined by three coordinated processes: macrophage iron recycling, duodenal iron absorption, and hepatic iron storage. When iron is administered therapeutically, it is assimilated by one or more of these three tissues, which play critical roles in iron metabolism and the maintenance of iron homeostasis (Fig. 8) (Andrews, 2000).

1. Macrophage iron recycling

Normal human erythrocytes have a finite life span of approximately 4 months. Tissue macrophages remove senescent and damaged erythrocytes from circulation and breakdown hemoglobin to recycle iron, supplying most of the requirement for new erythropoiesis. The process by which macrophages distinguish aged and damaged erythrocytes is not fully understood, but it is likely that morphological changes in the erythrocyte membrane facilitate recognition and uptake by macrophages (Knutson & Wessling-Resnick, 2003).

Binding of erythrocytes to the macrophage cell surface initiates phagocytosis and lysosome-mediated degradation of the erythrocyte membrane. Heme oxygenases catalyze the oxidation of heme to biliverdin, free iron, and carbon monoxide (Yachie, 1999).

Fig. 8 Major pathways of iron transfer between cells and tissues (Andrews, 2000).

Similar to the transferrin cycle, liberated iron may be pumped from the phagosome into the cytoplasm by DMT1, though this has not been definitively established. Heme-derived iron can be utilized by the cell, sequestered within the multimeric iron storage protein ferritin or exported into the plasma (Jabadoet al., 2002).

The transmembrane transporter ferroportin is activated in macrophages after erythrophagocytosis (Canonne-Hergauxet al., 2006). Ferroportin is the only cellular iron exporter that has been identified in vertebrates (Abboud & Haile, 2000). It is expressed in cells of the extraembryonic visceral endoderm that provide nourishment to the developing embryo, in the intestinal epithelium and in spleen and liver macrophages that recycle iron (Donovanet al., 2005).

Tissue macrophages contain large amounts of iron, apparently because they are unable to export it to the circulation. These results support the idea that most iron liberated from heme in macrophages is mobilized through ferroportin-mediated iron export to be utilized for erythropoiesis. Because ferroportin transports ferrous iron, iron must be oxidized to its ferric form in order to bind circulating transferrin (Harris, 1999).

A circulating ferroxidase, ceruloplasmin, is thought to carry out the oxidation of iron exported from macrophages also ceruloplasmin appears to be necessary to keep ferroportin on the cell surface. For both of these reasons, it is not surprising that ceruloplasmin deficiency (a ceruloplasminemia) leads to tissue iron loading with low transferrin saturation and, often, mild anemia (De Domenico, 2007).

2. Duodenal iron absorption

In contrast to some other metals, there is no regulated mechanism for iron excretion through the liver or kidneys. Macrophage-mediated iron recycling alone cannot sustain erythropoiesis over the long term. Early in life, the overall iron endowment must be increased to support growth. Later, small obligatory iron losses from bleeding and exfoliation of skin and mucosal cells would lead to negative iron balance if not offset by continuous iron intake. Thus, iron balance is achieved through regulated dietary iron absorption (Andrews, 2000).

Dietary iron absorption occurs in the most proximal part of the duodenum, the first section of the small intestine (Fig. 9). There, acidity from stomach acid aids in the absorption of both the heme iron, primarily derived from hemoglobin and myoglobin in meats and the inorganic iron, from other food sources (Qiuet al., 2007).

Inorganic iron in the intestinal lumen is primarily in its ferric, oxidized form. In order for iron to be absorbed, it must be reduced to the ferrous form. Reduction of iron can be carried out by an enterocyte apical membrane protein duodenal cytochrome b (Dcytb) (McKieet al., 2001).

The fact that the same iron transporter is used for cellular iron uptake both in transferrin cycle endosomes and at the apical surface of the intestinal epithelium is somewhat surprising. These two membranes are quite different and must be reached through distinct targeting signals. However, both sites are within a low-pH milieu, important because DMT1 uses cotransport of protons to move iron across the membrane (Sacheret al., 2001; Xuet al., 2004).

illustration not visible in this excerpt

Fig. 9 Iron absorption (Andrews, 2000).

Depending on body iron needs, intracellular iron can be stored within the enterocyte or exported into the plasma. Net absorption requires transfer across the basolateral surface of the epithelium—iron retained within enterocytes is lost from the body when those cells senesce and are shed into the gut lumen. It appears that the transmembrane transporter ferroportin is responsible for most if not all basolateral iron transfer (Donovanet al., 2005).

A ferroxidase activity acts in concert with ferroportin-mediated export. This can be supplied by the enterocyte-associated multicopper oxidase, hephaestin, or by its circulating homologue, ceruloplasmin (Vulpeet al., 1999).

3. Hepatocyte iron storage

Hepatocytes serve many important functions including detoxification of the blood production of proteins that aid in host defense and storage of essential nutrients such as glucose and iron. Excess circulating iron in both transferrin-bound and nontransferrin-bound forms can be taken up by hepatocytes (Trenoret al., 2000).

Similarly, DMT1 cannot account for all hepatocyte iron uptake because DMT1 knockout mice and human patients carrying loss-of-function mutations in DMT1 accumulate iron in the liver (Iolasconet al., 2006).

Other hepatic iron importers must exist to participate in efficient iron storage. Hepatocyte membrane proteins such as megalin, TFR2, CD163, and L-type calcium channels are candidate iron importers that employ a variety of molecular mechanisms to bring iron into cells (Borregaard & Cowland, 2006).

Although hepatocyte iron uptake is not fully understood, it is clear that once inside the cell, iron can be utilized in cellular processes or sequestered in ferritin. When iron loss or demand is too great to rely solely on recycling and absorption, iron is mobilized from hepatic storage to sustain erythropoiesis. Ferroportin and ceruloplasmin are believed to be involved in the process of iron export from hepatocytes, but this has not been shown directly (Kozyrakiet al., 2001; Kristiansenet al., 2001).

4. Systemic iron homeostasis

Systemic iron homeostasis maintains body iron content within tolerable limits and dictates iron distribution. As the largest consumer of iron, the erythron is particularly sensitive to iron insufficiency. When body iron stores are depleted, iron deficiency anemia ensues. Rarely, iron deficiency anemia can be caused by genetic lesions that interfere with intestinal iron absorption, erythroid iron assimilation, or both. The best characterized of these are mutations preventing the production of transferrin or inactivating DMT1 (Iolasconet al., 2006; Mimset al., 2004).

Far more commonly, deficiency results from an imbalance between increased iron requirements associated with growth and blood loss and iron acquisition from the diet. In this case, iron deficiency anemia is characterized by low plasma iron, decreased iron stores, and the accumulation of iron-free protoporphyrins. Iron-deficient erythrocytes are small (microcytic), pale (hypochromic), and relatively fragile. Oxygen- carrying capability caused by iron deficiency has measurable effects on quality of life, associated with symptoms including fatigue and tachycardia (Beutleret al., 2000).

Iron replacement therapy, through dietary supplementation, intravenous iron, or transfusion of iron-rich erythrocytes, is the only treatment for iron deficiency anemia. Although essential for several important cellular functions, iron’s capacity to donate and accept electrons makes the metal toxic. Several proteins such as transferrin, ferritin, lactoferrin, lipocalin, and myoglobin exist to bind iron in a variety of contexts. Binding to these proteins renders iron less able to react with its environment (Brittonet al., 2002).

Thus, a safe upper limit of body iron for each individual is set by the capacity of iron-binding proteins to sequester iron. Once this capacity is reached, free iron accumulates within the plasma and cells. The accumulation of excess iron is undesirable because ferrous iron reacts with hydrogen peroxide to produce hydroxyl radicals. These radicals damage macromolecules resulting in cellular and tissue dysfunction and organ failure associated with iron overload disorders (Brittonet al., 2002).

Iron Overload

The human body can only excrete one to two milligrams of iron. With every unit of blood, about 200 to 250 milligrams of iron are transfused, therefore iron overload will occur. Generally, iron overload can be classified as primary (hereditary) or secondary (acquired), depending on whether it results from a primary defect in the regulation of iron balance or secondary to other genetic or acquired disorders or their treatment:

Primary iron overload results from abnormally regulated iron absorption: Hereditary hemochromatosis is an example; caused by a missense mutation on the C282Y gene. This mutation results in the incorrect processing of regulatory receptors in the intestine (Porter, 2001).

Secondary iron overloadcan result as the indirect effect of a condition or occur as a result of its treatment:

Ineffective erythropoiesis and anemias requiring repeated blood transfusions such as (beta-thalassemia major, thalassemia intermediate, myelodysplastic syndromes and sickle cell disease) (Porter, 2001).

When iron homeostasis is in balance, iron is absorbed from the diet (gut) at a rate equivalent to 1-2 mg/day. After absorption from the duodenum, iron enters the plasma where it is complexed with transferrin. Transferrin-bound iron in the plasma is the main pool supplying iron to the erythron, which cycles 20-30 mg of iron each day, as well as to hepatocytes and other parenchyma, which cycle around 10% of this amount (Anderson et al.,2007).

When transferrin becomes completely saturated during conditions of iron overload, iron circulates in the bloodstream as extracellular non- transferrin-bound iron (NTBI). In the case of transfusion the reticuloendothelial system loses the capacity or gets filled up and has no ability to store more iron. It is bound to transferrin and when the capacity of transferrin is saturated, it becomes the non-transferrin bound iron (NTBI) (Andersonet al.,2007).

As iron loading progresses, the capacity of serum transferrin, the main transport protein of iron, to bind and detoxify iron may be exceeded and a non-transferrin- bound fraction of plasma iron may promote the generation of free hydroxyl radicals, propagators of oxygen-related damage. The body maintains a number of antioxidant mechanisms against damage induced by free radicals, including superoxide dismutases, catalase, and glutathione (Hershkoet al., 1998).

In the absence of chelating therapy the accumulation of iron results in progressive dysfunction of the heart, liver, and endocrine glands. Within the heart, changes associated with chronic anemia are usually present in patients who are not receiving transfusions and are aggravated by iron deposition. In response to iron loading, human myocytes in vitro increase the transport of non-transferrin-bound iron possibly thereby aggravating cardiac iron loading (Parkeset al., 1993).

Extensive iron deposits are associated with cardiac hypertrophy and dilatation, degeneration of myocardial fibers. In patients who are receiving transfusions but not chelating therapy, symptomatic cardiac disease has been reported within 10 years after the start of transfusions and may be aggravated by myocarditis and pulmonary hypertension (Aessoposet al., 1995; Duet al., 1997).

The survival of patients with β-thalassemia is determined by the magnitude of iron loading within the heart. Iron-induced liver disease is a common cause of death in older patients and is often aggravated by infection with hepatitis C virus. Within two years after the start of transfusions, collagen formation and portal fibrosis have been reported; in the absence of chelating therapy, cirrhosis may develop in the first decade of life. The risk of hepatic fibrosis is augmented at body iron burdens corresponding to hepatic iron concentrations of more than 7 mg per gram of liver, dry weight (Fig. 10) (Niederauet al., 1996).

Fig. 10 Hepatic Iron Burden over Time and the Effect of Various Hepatic Iron Concentrations in Patients with Thalassemia Major, Homozygous Hemochromatosis, and Heterozygous Hemochromatosis (Nancy & Livieri, 1999).

The transport of non-transferrin-bound iron is increased, possibly aggravating iron loading in vivo. Iron loading within the anterior pituitary is the primary cause of disturbed sexual maturation, early secondary amenorrhea occurs in approximately one quarter of female patients over the age of 15 years. Even in the modern era of iron-chelating therapy, diabetes mellitus is observed in about 5 percent of adults (Olivieriet al., 1998). As the iron burden increases and iron-related liver dysfunction progresses, hyperinsulinemia occurs as a result of reduced extraction of insulin by the liver, leading to exhaustion of beta cells and reduced circulating insulin concentrations. Studies reporting reduced serum concentrations of trypsin and lipase suggest that the exocrine pancreas is also damaged by iron loading. Over the long term, iron deposition also damages the thyroid, parathyroid, adrenal glands and may provoke pulmonary hypertension, right ventricular dilatation, and restrictive lung disease. Bone density is markedly reduced in patients with β-thalassemia (Taiet al., 1996; Olivieriet al., 1998).

Iron Chelation Therapy

Indications for chelation therapy

(i) Transfusions of 2 units/month persisting for at least one year.
(ii) Ferritin level of 1000 ng/ml.
(iii) Patients in which transplant is imminent.
(iv) Consider earlier chelation therapy in patients with compromised organ function who experience increased transfusion burden (Franchini&Veneri 2004).

Treatment Options

Three products are available worldwide:

1)Deferoxamine:

Deferoxamine is indicated for the treatment of acute iron intoxication and of chronic iron overload due to transfusion-dependent anemias. Deferoxamine chelates iron by forming a stable complex that prevents the iron from entering into further chemical reactions. It readily chelates iron from ferritin and hemosiderin but not readily from transferrin; it does not combine with the iron from cytochromes and hemoglobin. Deferoxamine does not cause any demonstrable increase in the excretion of electrolytes or trace metals. Theoretically, 100 parts by weight of Deferoxamine is capable of binding approximately 8.5 parts by weight of ferric iron (Borgna- Pignattiet al,2004).

Deferoxamine is metabolized principally by plasma enzymes, but the pathways have not yet been defined. The chelate is readily soluble in water and passes easily through the kidney, giving the urine a characteristic reddish color. Some is also excreted in the feces via the bile. Long-term therapy with deferoxamine slows accumulation of hepatic iron and retards or eliminates progression of hepatic fibrosis. Iron mobilization with deferoxamine is relatively poor in patients under the age of 3 years with relatively little iron overload. The drug should ordinarily not be given to such patients unless significant iron mobilization (e.g., 1 mg or more of iron per day) can be demonstrated (Borgna-Pignattiet al,2004).

2)Deferiprone:

Promising chelating compounds are the 3-hydroxypyrid- 4-ones which form strong, highly stable and water soluble 3:1 complexes with the Fe3+- ion at physiological pH both in vitro and in vivo. The binding constants are high in comparison with those of desferrioxamine and transferrin, the physiological transport protein for Iron. They are capable of mobilizing iron from transferrin, ferritin, hemosiderin, hepatocytes and macrophages .The affinity for divalent metal ions is low (Franchini&Veneri 2004).

The first representative of this group which has been tested in humans is deferiprone. This compound has shown very little toxicity in animal studies.

After oral ingestion, deferiprone is rapidly absorbed, metabolised in the liver and excreted in the urine as glucuronide (at least 90% of the absorbed dose), as iron complex or as unchanged drug .Relatively little is known about the pharmacodynamics of deferiprone (Franchini&Veneri 2004).

3) Deferasirox:

Exjade (deferasirox) is an orally active chelator that is selective for iron (as Fe3+). It is a tridentate ligand that binds iron with high affinity in a 2:1 ratio. Although deferasirox has very low affinity for zinc and copper there are variable decreases in the serum concentration of these trace metals after the administration of deferasirox. The clinical significance of these decreases is uncertain. Deferasirox is highly (~99%) protein bound almost exclusively to serum albumin. The percentage of deferasirox confined to the blood cells was 5% in humans. The volume of distribution at steady state (Vss) of deferasirox is 14.37 ± 2.69 L in adults. Deferasirox and metabolites are primarily (84% of the dose) excreted in the feces. Renal excretion of deferasirox and metabolites is minimal (8% of the administered dose). The mean elimination half-life (t1/2) ranged from 8-16 hours following oral administration (Cappelliniet al., 2006).

HEPCIDIN

Hepcidin is the key regulator of systemic iron homeostasis and a pathogenic factor in anemia of inflammation and hereditary hemochromatosis. Hepcidin inhibits iron influx into plasma from duodenal enterocytes that absorb dietary iron, from macrophages that recycle iron from senescent erythrocytes and from hepatocytes that store iron. Hepcidin acts by binding to the cellular iron exporter ferroportin and causing its internalization and degradation. Hepcidin production is increased by iron excess and inflammation and decreased by anemia and hypoxia, however, the molecular mechanisms of hepcidin regulation by iron, oxygen and anemia are still unclear. Iron-loading anemias are disorders in which hepcidin is regulated by opposing influences of ineffective erythropoiesis and concomitant iron overload (Pigeonet al.,2001).

Hepcidin peptide:

Hepcidin is a 25-amino-acid iron peptide hormone. Initially identified in human plasma and urine as an anti-microbial molecule. located on chromosome 19q13.1, encodes a precursor protein preprohepcidin of 84 amino acids (aa). During its export from the cytoplasm, this full-length pre- prohepcidin undergoes enzymatic cleavage, resulting in the export of a 64 aa prohepcidin peptide into the ER lumen. Serum prohepcidin levels have been widely used to evaluate iron overload in clinical and preclinical studies (Parket al.,2001).

Bioactive hepcidin indeed bears structural similarity to disulfide-rich antimicrobial peptides. Hepcidin is synthesized in the liver as a propeptide and has a characteristic furin cleavage site immediately N-terminal to the 25-amino-acid peptide (Parket al.,2001).

The molecule is a simple hairpin whose 2 arms are linked across by disulfide bridges in a ladderlike configuration. One highly unusual feature of the molecule is the presence of disulfide linkage between 2 adjacent cysteines near the turn of the hairpin. Compared with most disulfide bonds, disulfide bonds formed between adjacent cysteines are stressed and could have a greater chemical reactivity. Like other antimicrobial peptides, hepcidin displays spatial separation of its positively charged hydrophilic side chains from the hydrophobic ones, a characteristic of peptides that disrupt bacterial membranes (Fig. 11) (Detivaudet al.,2005).

illustration not visible in this excerpt

Fig. 11 Amino acid sequence and a model of the major form of human hepcidin. The amino and carboxy termini are labeled as N and C, The pattern of disulfide linkages between the 8 cysteines is also shown in the amino acid sequence (Ganz, 2003).

Molecular regulation of hepcidin

The expression of hepcidin is regulated by many genes as HFE, Hemojuvelin (HJV) and growth differentiation factor 15 (GDF15) (Papanikolaouet al., 2004) .

HFE, Human hemochromatosis protein an MHC-class 1-like molecule, is highly expressed in crypt cells (enterocyte, macrophage and hepatocyte). The HFE gene is located on short arm of chromosome 6 at location 6p21.3. It seems to enhance transferrin-bound iron uptake from the plasma into crypt cells via TfR1, and may also inhibit the release of iron from the cell via ferroportin. It regulates hepcidin expression, mechanism is uncertain but it may participate in a signaling complex with TfR2 and interacts with TfR1 & β-2-microglobulin (Munozet al.,2009).

Hemojuvelin (HJV) is a membrane-bound and soluble protein in mammals that is responsible for the iron overload condition known as juvenile hemochromatosis in humans, a severe form of hemochromatosis. The hemojuvelin protein is encoded by the HFE2 gene. Mutations in HJV are responsible for the vast majority of juvenile hemochromatosis patients. Hemojuvelin is highly expressed in skeletal muscle and heart, and to a lesser extent in the liver. One insight into the pathogenesis of juvenile hemochromatosis is that patients have low to undetectable urinary hepcidin levels, suggesting that hemojuvelin is a positive regulator of hepcidin, the central iron regulatory hormone (Papanikolaouet al., 2004).

For many years the signal transduction pathways that regulate systemic iron homeostasis have been unknown. However, a study by Babitt et al 2006 suggested that hemojuvelin interacts with bone morphogenetic protein (BMP), possibly as a co-receptor, and may signal via the SMAD pathway to regulate hepcidin expression (Zhanget al.,2008).

Recently, the iron and erythropoiesis-controlled growth differentiation factor 15 (GDF15) has been shown to inhibit the expression of hepcidin in β-thalassaemia patients, thereby increasing iron absorption despite iron overload. (Tamaryet al.,2008).

Mechanism of hepcidin action

Hepcidin causes a decrease in serum iron. Injection of hepcidin agonist into mice results in hypoferremia already within 1 hour, and a similar effect was seen with acute induction of hepcidin expression in tetracyclineinducible transgenic mice. The hypoferremia develops because hepcidin blocks the supply of iron into plasma while the relatively small plasma iron pool is rapidly used up by erythrocyte precursors. Hepcidin blocks iron flows from macrophages recycling iron, from stores in the liver and from enterocytes absorbing dietary iron (Viatteet al.,2005).

The molecular mechanism is based on hepcidin's interaction with ferroportin. Ferroportin is the only known cellular iron exporter in vertebrates, and is expressed in all the tissues handling major iron flows as reticulo-endothelial macrophages, hepatocytes and duodenal enterocytes. Hepcidin binds to ferroportin and causes its internalization and degradation in lysosomes, thus effectively blocking the export of iron from the cells (Nemethet al.,2004).

In vitro, the internalization of ferroportin occurs less than 1 hour after addition of hepcidin, consistent with the kinetics of hypoferremia observed in vivo. Likewise, injection of radiolabeled hepcidin in mice resulted in equally rapid accumulation of radioactive hepcidin in ferroportin-rich organs (spleen, duodenum and liver), providing further support for the key role of hepcidin-ferroportin interactions in the regulation of iron transport (Fig. 12) (Riveraet al.,2005b).

illustration not visible in this excerpt

Fig. 12 Physiology of hepcidin-ferroportin interaction (Rivera et al., 2005b).

- Ferroportin = iron export protein.
- Circulating hepcidin.
- Hepcidin binds to ferroportin.
- Internalization, then ferroportin degradation.
- Degraded ferroportin.
- Decreased iron release due to decreased ferroportin.

Hepcidin maintains iron homeostasis through a physical interaction with ferroportin has led to a plausible model for the normal maintenance of iron homeostasis (Fig. 13) and the disruption of homeostasis in human disease (Zoller&Cox 2005).

illustration not visible in this excerpt

Fig. 13 Normal iron homeostasis mediated by an iron-sensing feedback loop (Wrighting & Andrews, 2008).

Hepcidin is the principal regulator of extracellular iron concentration

Hepcidin is increased by iron loading and this provides the homeostatic loop to maintain normal extracellular concentrations of iron. A rise in plasma iron (e.g. after a meal or an iron supplement) leads to increased hepcidin production. In turn, elevated hepcidin reduces the concentration of ferroportin molecules on the cell surface and inhibits the entry of iron into plasma, thus allowing the iron concentration to return to normal levels. Conversely, in iron deficiency, hepcidin production decreases, allowing a greater export of iron through ferroportin into plasma; this results in an appropriate rise in circulating iron (Delabyetal.,2005).

Chronic alterations of hepcidin expression result in systemic disorders of iron metabolism and maldistribution of iron in the body. Homozygous disruption of the hepcidin gene in humans or mice leads to severe iron overload. Conversely, overexpression of hepcidin in transgenic mice resulted in severe microcytic, hypochromic anemia. Mice with tumor xenografts engineered to overexpress hepcidin also developed hypoferremia and anemia, with iron sequestration in the stores (Riveraet al.,2005a).

Similarly, overproduction of hepcidin by liver tumors in patients with type 1a glycogen storage disease caused iron-refractory anemia which resolved only after resection of the tumor, or after liver transplantation. Altogether, these studies confirm the role of hepcidin as the negative regulator of iron absorption, recycling and release from stores (Weinsteinet al.,2002).

Regulation of hepcidin synthesis; implications for the disorders of iron metabolism As an iron-regulatory hormone, hepcidin synthesis is increased by iron loading and inflammation and is decreased by anemia and hypoxia. Except for inflammation, the molecular pathways underlying regulation of hepcidin are not well understood. Dysregulation of hepcidin synthesis, however, appears to be the key factor in the pathogenesis of a spectrum of iron disorders, with hepcidin deficiency causing iron overload and elevated hepcidin mediating anemia of inflammation (Lorealet al.,2005).

Regulation by inflammation

Hepcidin synthesis is markedly induced by infection and inflammation. In animal models, injection of turpentine, lipopolysaccharide, or Freund's adjuvant increased hepatic hepcidin mRNA expression, and in humans, infusion of lipopolysaccharide resulted in a rapid increase in urinary hepcidin. These effects are mediated by inflammatory cytokines including interleukin (IL)-6 and IL-1. IL-6 is sufficient for hepcidin induction since direct treatment of primary hepatocytes with IL-6 resulted in rapid upregulation of hepcidin mRNA and infusion of human volunteers with IL- 6 resulted in increased urinary hepcidin excretion within just 2 hours after infusion (Nemethet al.,2004 ; Nemethet al.,2003).

Hepcidin as a mediator of anemia of inflammation

Hepcidin increase was associated with hypoferremia in all the inflammatory models. Increased hepcidin appears to be the key factor in the development of anemia of inflammation. Hypoferremia and anemia of inflammation have likely developed during evolution as a host defense strategy against infection, limiting the growth of invading microbes. However, the same strategy has become maladaptive with the increasing incidence of non-infectious diseases associated with excessive cytokine production, including rheumatologic diseases, inflammatory bowel disease, multiple myeloma and other malignancies (Riveraet al.,2005a).

Anemia of inflammation is characterized by decreased serum iron and impaired mobilization of iron from stores, evident from the presence of iron in bone-marrow macrophages and increased ferritin levels. These are the very features observed in mouse models with increased hepcidin. Intraperitoneal injection of synthetic hepcidin resulted in hypoferremia within 1 hour, and chronic over expression of hepcidin in tumors resulted in anemia and hypoferremia despite increased liver iron stores (Riveraet al., 2005b).

Furthermore, patients with infection or inflammatory disorders have elevated urinary excretion of hepcidin compared to healthy controls. Thus, the molecular pathway from inflammation to anemia centers on the elevated plasma hepcidin which causes the internalization and degradation of ferroportin in macrophages, hepatocytes and duodenal enterocytes, sequestering iron in these cells and blocking iron flows into plasma (Nemethet al.,2003).

As the bone marrow continues to utilize iron for hemoglobin synthesis, the small plasma iron compartment becomes rapidly depleted causing hypoferremia. Persistent hypoferremia, as in chronic inflammation, leads to iron-restricted erythropoiesis and anemia. However, it still remains to be established whether the increase in hepcidin is the essential factor in the development of this disorder, since inflammation may contribute to anemia by alternative hepcidin-independent mechanisms including decreased erythropoietin production, blunted response to erythropoietin and shortened erythrocyte lifespan (Nemethet al.,2003).

Regulation of hepcidin by anemia and hypoxia

Inadequate delivery of oxygen to tissues, which occurs in anemia or hypoxemia, would be expected to result in homeostatic decrease in hepcidin synthesis. The decrease in hepcidin levels would in turn allow increased iron mobilization from macrophages and hepatocytes, and increased iron absorption from the diet, making more iron available for erythrocyte production. Indeed, hepcidin was shown to be suppressed by anemia and hypoxia; however, the molecular pathways that regulate this response are still unclear. Though anemia may act by causing liver hypoxia, it is also possible that the pathways of hepcidin regulation by oxygen and by anemia/erythropoiesis are independent (Nicolaset al.,2002).

illustration not visible in this excerpt

Fig. 14 Hepcidin mRNA expression (Nicolaset al.,2002).

Exposure to hypoxia decreased hepcidin mRNA expression (Fig. 14). In general, cellular oxygen sensing and the related transcriptional control are largely mediated by the hypoxia-inducible factor (HIF). However, unlike most of the target genes that are transcriptionally activated by HIF, hepcidin expression is negatively regulated by hypoxia. In addition, except for human hepcidin promoter, the promoters in other mammals do not contain the consensus binding sites for HIF, and direct involvement of HIF in transcriptional regulation of hepcidin remains to be explored (Nicolaset al., 2002).

Hepcidin mRNA increased by dietary iron and mice injected with bacterial LPS (indirect effect mediated by IL-6). Decreased by Hypoxemia and increased erythroid iron demand (phlebotomy or hemolysis).

Hepcidin and anemia of chronic disease

Hepcidin appears to block iron uptake in the duodenum and release from RE macrophages, thereby decreasing delivery of iron to RBC precursors. A hepcidin antagonist might benefit patients with ACD but might be contraindicated in patients with infections . Elevated urinary hepcidin levels might be useful to diagnose ACD (Nemethet al.,2003).

Role of Hepcidin in Iron -Loading Anemias

Iron-Loading anemias are characterized by ineffective erythropoiesis and increased intestinal iron absorption. Erythrocyte transfusions further exacerbate the iron overload, the development of hepcidin- based diagnostics and therapies for iron-loading anemias may offer more effective approaches to prevent the toxicity associated with iron overload. The most common iron- loading anemias are the intermediate and major forms of β- thalassemia, but other rare anemias also complicated by iron loading, including congenital dyserythropoietic anemia, X- Linked sideroblastic anemia and anemia associated with divalent metal ion transporter 1 (DMT1) mutations (Papanikolaouet al., 2005) .

Role of Hepcidin in β-thalassemia major

In the presence of systemic iron overload, patients with thalassemia major in whom iron overload was more severe and anemia was partially relieved by transfusions, had urinary hepcidin concentrations that were higher than in thalassemia intermedia. These findings were interpreted as supporting the dominant erythropoietic effect of exogenous hepcidin could prevent the iron overload in iron-Loading anemias (Lorealet al., 2005).

Iron overload of tissue is the most important complication of β - thalassemia major and is a major focus of management. In patients who are not receiving transfusions, abnormally regulated iron absorption results in increases in body iron burden ranging from 2 to 5 g per year, depending on the severity of erythroid expansion. Regular transfusions may double this rate of iron accumulation (Lorealet al., 2005).

Elevated levels of hepcidin in the bloodstream effectively shut off iron absorption by disabling the iron exporter ferroportin. Conversely, low levels of circulating hepcidin allow ferroportin to export iron into the bloodstream. Aberrations in hepcidin expression result in disorders of iron deficiency and iron overload. It is clear that erythroid precursors communicate their iron needs to the liver to influence the production of hepcidin and thus the amount of iron (Lorealet al., 2005).

In a mouse model of beta-thalassaemia, Weizer-Stern and his co- authors (2006) observed that the liver expressed relatively low levels of hepcidin, which is a key factor in the regulation of iron absorption by the gut and of iron recycling by the reticuloendothelial system. It was hypothesised that, despite the overt iron overload, a putative plasma factor found in beta-thalassaemia might suppress liver hepcidin expression. Sera from beta-thalassaemia patients were compared with those of healthy individuals regarding their capacity to induce changes in the expression of key genes of iron metabolism in human HepG2 hepatoma cells. Sera from beta-thalassaemia major patients induced a major decrease in hepcidin (HAMP) expression. A significant correlation was found between the degree of downregulation of HAMP induced by beta-thalassaemia major sera.

Adamsky and his co-authors (2004) have found that iron overload is less dominant than anaemia in regulating hepcidin expression in the setting of the β-thalassemia major mouse model. The decreased expression of hepcidin may explain the increased absorption of iron in thalassemia. Recently, decreased expression of hepcidin was found in hereditary haemochromatosis in association with elevated levels of nontransferrin bound iron. The elevated expression of NGAL, an alternative iron delivery vehicle, supports the role of nontranferrin bound iron in the abnormal iron regulation in thalassemia. The decreased HFE expression level is similar to the finding in hereditary haemochromatosis (Bridleet al., 2003).

Regulation of hepcidin by iron and the lessons from hereditary hemochromatosis

The only clues about molecules involved in the pathway of hepcidin regulation by iron come from mutations causing hereditary hemochromatosis. In addition to juvenile hemochromatosis caused by inactivating mutations in the hepcidin gene itself, it appears that hepcidin deficiency is the unifying cause of most types of hereditary hemochromatosis. Measurements of urinary hepcidin excretion or hepatic mRNA expression showed that patients and animal models with homozygous disruption of HFE, transferrin receptor 2 (TfR2) and hemojuvelin (HJV) all had hepcidin levels inappropriately low for the systemic iron load (Muckenthaleret al.,2003; Ahmadet al.,2002).

While the precise function of the three molecules is not known, they likely participate in the sensing of iron or the consequent signal transduction that regulates hepcidin synthesis and release. Importantly, the degree of hepcidin deficiency appears to correlate with the severity of the disease. The most severe form, juvenile hemochromatosis, is caused by mutations in either the hepcidin or HJV gene and these are phenotypically undistinguishable. Patients with HJV mutations have very low or undetectable urinary hepcidin suggesting that HJV is the key regulator of hepcidin (Papanikolaouet al.,2004).

Genetic iron-overload disorders may be divided into haemochromatotic and non-haemochromatotic forms according to patho-physiological and phenotypic criteria (Table 1) (Pietrangelo, 2007). Haemochromatosis refers to hereditary iron-overload disorders characterized by normal erythropoiesis, increased transferrin saturation and parenchymal distribution of iron deposition, and related to an inaccurate production and/or regulation and/or activity of hepcidin (Pietrangelo, 2007).

Hepcidin and Erythropoeisis

Hepcidin expression is regulated in response to bone marrow needs

Iron absorption is increased in patients with congenital anemias characterized by ineffective erythropoiesis. Clinically, increased intestinal iron absorption compounds the effects of transfusional iron overload in patients with thalassemia syndromes, sideroblastic anemia, or congenital dyserythropoietic anemias (Adamskyet al., 2004).

Finch (1994) proposed the existence of an erythroid regulator of systemic iron homeostasis. The erythron, composed of developing erythroid cells in the bone marrow and circulating erythrocytes, utilizes about 80% of the iron found in the plasma. Anemia results from the inability of the erythroid compartment to receive its full complement of iron. The putative erythroid regulator communicates the iron needs of the erythron to influence changes in intestinal iron absorption (Bredaet al., 2005).

Table 1 Main characteristics of genetic iron overload disorders (Deugnieret al., 2008).

illustration not visible in this excerpt

Hepcidin is an effective inhibitor of iron absorption, the erythroid regulator includes a mechanism to decrease hepcidin production. Accordingly, low hepcidin levels have been reported with thalassemia and other disorders with ineffective erythropoiesis. In these disorders, decreased hepcidin expression leads to relief of inhibition of ferroportin, resulting in increased iron release from recycling macrophages and absorptive enterocytes, increasing availability of iron for erythropoiesis. However, the iron cannot be effectively utilized by the erythron, leading to accumulation and tissue iron overload in the face of anemia (Gardenghiet al., 2007; Jenkinset al., 2007).

Stimulation of erythropoiesis with phenylhydrazine resulted in hepcidin suppression as expected, but the simultaneous inhibition of erythropoiesis by irradiation prevented hepcidin suppression despite severe anemia. In addition, irradiation prevented hepcidin suppression after erythropoietin administration, ruling out the direct effect of erythropoietin on hepcidin synthesis (Bredaet al.,2005).

SUBJECTS AND METHODS

This study was carried out in pediatric and clinical pathology departments of Zagazig University Hospitals in the period from March 2009 to April 2010. The study included 40 children divided into two groups:

I- Group (I): healthy controls group

This group included (10) apparently healthy subjects aged between 4-11 years with a male to female ratio of 1.5:1 (6 males and 4 females).

II- Group (II): patients group

The studied group included 30 β-thalassemic major children patients aged between 4-10 years with a male to female ratio of 1.5:1 (18 males and 12 females). They were selected from patients attending pediatric hematology unit and already diagnosed as β-thalassemia major. All study members were subjected to the following:

a) Full history taking to collect data from children or mothers: Personal history, diagnosis, blood transfusion, iron chelation and history of complications (hepatic, renal, cardiac and endocrinal complications).

b) Clinical examination included:

1) General examination

2) Local examination

- Head and neck examination for mongoloid face, earthy face of poorly chelated patient.
- Cardiac examination to assess cardiac complications.
- Abdominal examination to assess spleen and liver state (splenomegally, splenectomy or hepatomegally).
- Abnormalities of long bones and skull. Laboratory investigations:

A-Routine:

(1) C.B.C by (SYSMEX SF 3000).
(2) Serum iron (colorimetric) (Hueberset al., 1987).
(3) Serum ferritin (Cobas E 411).
(4) TIBC( colorimetric ) (Finch & Huebers, 1986)
(5) Hemoglobin electrophoresis (Valeriet al., 1965).
(6) Liver function tests, kidney function tests and serum Alkaline phosphatase test were done by (Selectra XL).

B- Special investigations

Hepcidin hormone:

Hepcidin was measured by Enzyme Linked Immunosorbant Assay in the serum (Park et al.,2001). The Kit was supplied from DRG International,Inc.,USA.

The principle:

The DRG Hepcidin ELISA Kit is a solid phase enzyme-linked immunosorbent assay, based on the principle of competitive binding. The microtiter wells are coated with a monoclonal antibody directed towards the antigenic site of the bioactive Hepcidin 25 molecule. Endogenous Hepcidin of a patient sample competes with the added Hepcidin-biotin conjugate for binding to the coated antibody. After incubation the unbound conjugate is washed off. An incubation with a streptavidin-peroxidase enzyme complex and a second wash step follows. The addition of substrate solution results in a colour development which is stopped after a short incubation The intensity of colour developed is reverse proportional to the concentration of Hepcidin in the patient sample.

The reagents:

The kit reagents include:

(a) Standard: concentrations of synthetic pepide Hepcidin.
(b) Control low and high.
(c) Assay Buffer.
(d) Enzyme Complex contains : Streptavidin peroxidase.
(e) Substrate Solution contains:Tetramethylbenzidine(TMB).
(f) Stop Solution contains: H2SO4.
(g) Wash Solution (40X concentrated).

Reagent Preparation:

- All reagents and required number of strips were brought to room temperature prior to use.
- The lyophilized contents of the standard vials were reconstituted with 0.5 mL Aqua dest.
- The lyophilized content of the control was reconstituted with 0.5 mL Aqua dest. And was left to stand for 10 minutes in minimum.
- Deionized water was added to the 40X concentrated Wash Solution.

30 mL of concentrated Wash Solution was diluted with 1170 mL deionized water to a final volume of 1200 mL.

Specimen collection

Specimens have been prepared by collecting blood by clean venipuncture, allowed to clot, and serum has been separated by centrifugation at 2500 x g for 10 min at 4oC.Specimens were frozen until use at -20oC.

Assay Procedure

- The desired numbers of micro-titer wells were secured in the holder.
- 10 µl of Sample Buffer was dispensed into each well.
- 20 µl of each Standard, Control and Sample with new disposable tips were dispensed into appropriate wells.
- Incubation for 30 minutes at room temperature on a plate shaker at ≈ 500 rpm was done.
- 150 μL of Assay Buffer and 100 μL of Enzyme Conjugate were added to each well.
- Incubation for 180 minutes at room temperature on a plate shaker at ≈ 500 rpm was done.
- The contents of the wells were briskly shaked out then the wells were Rinsed 5 times with diluted wash solution (400 μL per well) and were Striked sharply on absorbent paper to remove residual droplets .
- 100 μL of Enzyme Complex was dispensed into each well.
- Incubation for 45 minutes at room temperature was done.
- The contents of the wells were briskly shaked out then the wells were Rinsed 5 times with diluted wash solution (400 μL per well) and were Striked sharply on absorbent paper to remove residual droplets.
- 100 μL of substrate solution was added to each well.
- Incubate for 30 minutes at room temperature was done.
- The enzymatic reaction was stopped by adding 100 µl of Stop Solution to each well.

After adding the Stop Solution, the OD at 450±10 nm was red with a microtiter plate reader within 10 minutes.

Calculation of Results

Calculations were done as follows: The average absorbance values for each set of standards, controls and patient samples were calculated. A standard curve was constructed by plotting the mean absorbance obtained from each standard against its concentration. The mean absorbance value for each sample was used to determine the corresponding concentration .

As the value of iron absorbance increases, the concentration of hepcidin in serum decreases and vice versa (i.e., reverse proportion relationship). The concentration of hepcidin in serum was calculated using the curve fitting equation and shown in Fig. 15.

illustration not visible in this excerpt

Fig. 15 Concentration of hepcidin of patients (in circles) and concentration of hecidin of control (in squares).

Statistical analysis

Data were entered checked and analyzed Epi-Info version 6 and SPP for windows version 11 (Dean et al., 1994). SPSS windows version 11 was used for data analysis as follows:

A- Descriptive statistics:

In which data were summarized using:

1) The arithmetic mean (X) as an average describing the central tendency of observations was applied.
2) The standard deviation (SD) as a measure of dispersion of the results around the mean was calculated.

B- Comparison of means:

The comparison was done using the student "t" test for comparison of means of two independent groups.

C- Correlation study:

Correlation between variables was done using correlation coefficient "r".

This test detects if the change in one variable was accompanied by a corresponding change in the other variable or not. The value ofrusually lies between -1 and +1, where positive indicate a tendency for X and Y to increase together while negative values indicate a tendency for X to increase with decrease of Y and vice versa. The significance of ‘r’ was obtained from the ‘t’ distribution with (n-2) degrees of freedom wherenis number of observation in each group .

D- Level of significance:

For all above mentioned statistical tests done, the threshold of significance is fixed at 5% level (P value), where:

a) P-value > 0.05 indicates non- significant results.
b) P-value < 0.05 indicates significant results.
c) P-value < 0.001 indicates highly significant results.

RESULTS

This study included 40 subjects divided into 10 normal control subjects (Group

I) and 30 β thalassemia major patients (Group II). The age of the normal subjects in Group I ranged from 5 to 11 years and that of the patients in Group II ranged from 4 to 10 years as represented in Table 2:

Table 2: Clinical data of the studied groups.

illustration not visible in this excerpt

Table 3: Liver and kidney functions results of the studied groups.

illustration not visible in this excerpt

Table (3) showed that there was a highly significant elevation of Total Bilirubin , Direct Bilirubinin , SGPT, SGOT and Alkaline phosphatase in patient group compared to control group (P < 0.001). However there was no significant difference for Total protein , S. Albumin , blood urea, S. Creatinine and BUN between the two groups.

Table 4 Complete blood count results of the studied groups.

illustration not visible in this excerpt

Table (4) and Fig. 16 showed that there was a highly significant reduction in group II compared to group I as regard RBCs, HCT, HB, MCV, MCHC and PDW (P < 0.001). However there was a significant elevation for WBC in group II compared to group I (P < 0.003) . No significant difference for RDW, PLT and MPV tests.

illustration not visible in this excerpt

Fig. 16 Variations of RBCs, HB, MCV, HCT (PCV) for 40 patients (30 patients and 10 controls).

Table 5: Results of iron study of the studied groups.

illustration not visible in this excerpt

Table (5) showed that there was a highly significant difference between group I and group II for Serum Iron, Serum Ferritin and TIBC, (P < 0.001).

Table 6: Hemoglobin Electrophoresis data of the studied groups.

illustration not visible in this excerpt

Table (6) showed that there was a highly significant difference between the two groups for HBA, HBA2 and HBF, (P < 0.001).

Table 7: Hepcidin concentration levels of the studied groups.

illustration not visible in this excerpt

Table (7) and Fig.17 showed that there was a highly significant reduction of hepcidin in Group (II) compared to Group (I) (p < 0.001)

illustration not visible in this excerpt

Fig. 17 Hepcidin and Serum Ferritin for 40 patients (30 patients and 10 controls).

Table 8: Ratio between hepcidin and Serum Ferritin of the studied groups..

illustration not visible in this excerpt

Table (8) showed that there was highly significant increase as regard hepcidin / Serum Ferritin ratio in Group (I) compared to Group (II). The hepcidin / Serum Ferritin ratio in the patients group was markedly reduced.

Table 9: Correlation between hepcidin and other parameters of the studied groups.

illustration not visible in this excerpt

Table (9) and Fig. 18-21 showed that there was a positive correlation between hepcidin in one side and Hb, Hct and Mcv in the other side (p<0.001), (Figs. 18- 20). While there was a negative (i.e., inverse) correlation between hepcidin and (ferritin &Serum Iron) (P< 0.001) (see Fig. 21). There was a positive correlation correlation between hepcidin in one side and TIBC (p<0.001) in the other side.

illustration not visible in this excerpt

Fig. 18 Correlation between hepcidin and Hb.

illustration not visible in this excerpt

Fig. 19 Correlation between hepcidin and HCT.

illustration not visible in this excerpt

Fig. 20 Correlation between hepcidin and MCV.

illustration not visible in this excerpt

Fig. 21 Correlation between hepcidin and Serium Ferritin.

DISCUSSION

β-thalassemia is the most common chronic hemolytic anemia in Egypt (85.1%). A carrier rate of 9-10.2% has been estimated in 1000 normal random subjects from different geographical areas of Egypt. β-thalassemia is much more common in Mediterranean countries constituting a major public health problem (El-Beshlawy, 1999).

Finch (1994) proposed the existence of an erythroid regulator of systemic iron homeostasis. The erythron, composed of developing erythroid cells in the bone marrow and circulating erythrocytes, utilizes about 80% of the iron found in the plasma. Anemia results from the inability of the erythroid compartment to receive its full complement of iron. The putative erythroid regulator communicates the iron needs of the erythron to influence changes in intestinal iron absorption (Bredaet al.,2005).

Iron absorption is increased in patients with congenital anemias characterized by ineffective erythropoiesis. Clinically, increased intestinal iron absorption compounds the effects of transfusional iron overload in patients with thalassemia syndromes (Adamsky et al., 2004).

The most common secondary complications are those related to transfusional iron overload, which can be prevented by adequate iron chelation. Iron-loading anemias are disorders in which hepcidin is regulated by opposing influences of ineffective erythropoiesis and concomitant iron overload (Pigeon et al., 2001).

Hepcidin is an effective inhibitor of iron absorption; the erythroid regulator includes a mechanism to decrease hepcidin production. Accordingly, low hepcidin levels have been reported with thalassemia and other disorders with ineffective erythropoiesis. In these disorders, decreased hepcidin expression leads to relief of inhibition of ferroportin, resulting in increased iron release from recycling macrophages and absorptive enterocytes, increasing availability of iron for erythropoiesis. However, the iron cannot be effectively utilized by the erythron, leading to accumulation and tissue iron overload in the face of anemia (Gardenghiet al.,2007 and Jenkinset al.,2007).

This study aim was to measure hepcidin concentration in patients of β thalassemic major to explain its role in iron metabolism for these patients who have iron overload.

This study revealed a statistically significant elevation of total Bilirubin and Direct Bilirubin (TB-DB) in the patient group compared to the control group, on the other hand there was no significant difference in the Total protein and S. Albumin. There was a statistically significant elevation in the patient group as regard SGPT, SGOT and Alkaline phosphatase, compared to the control group.

Hyperbillirubinemia is a result of chronic Hemolysis and ineffective erythropoiesis. Which together cause the anemia that occurs in thalassemia. The relative contributions of these two pathologic processes differ in various forms of thalassemia. The bone marrow of patients with thalassemia contains five to six times the number of erythroid precursors as does the bone marrow of healthy controls with 15 times the number of apoptotic cells in the polychromatophilic and orthochromic stages (Centiset al., 2000;Mathiaset al.,2000).

Ineffective erythropoiesis the major cause of accelerated apoptosis, is caused by excess -chain deposition in erythroid precursors. Although the exact mechanism is not known, a death-receptor-mediated pathway seems to be involved with Fas-Fas ligand interactions (De Mariaet al.,1999).

In normal erythropoiesis, apoptotic mechanisms seem to play a regulatory role and are required for normal erythroid maturation (Testa, 2004). Accelerated apoptosis is associated with a rise in extracellular exposure of phosphatidylserine, an important signal for removal by activated macrophages, whose numbers are increased in thalassemic bone marrow erythrocytes , resulting in their accelerated peripheral destruction ( Angelucciet al., 2002).

In this work there was significant reduction in the patient group as regard RBCs, HCT, HB, MCH, , MCV and MCHC. On the other hand, the WBC count was elevated in β thalassemia major patient group.

Leukocytosis is usually present, even after excluding the nucleated RBCs. A shift to the left is also encountered, reflecting the hemolytic process. The anemia is due to a combination of ineffective erythropoiesis, excessive peripheral red blood cell hemolysis, and progressive splenomegaly. The latter causes an increase in plasma volume and a decrease in total red cell mass. The red cells are microcytic (mean corpuscular volume <70 fL) with marked anisochromasia. Hypochromic microcytic anemia lead to MCV and MCH are significantly low, low MCHc, low HCT and low Hb (Wonke, 2001).

In this study there was a highly significant difference between the two groups for HBA, HBA2 and HBF, as HbA is the major hemoglobin found in adults and children. Hb A2 and HbF are found in small quantities in adult life but in β thalassemia major there was elevated HbF and Hb A2 but HbA is in small quantities (Rachmilewitz&Schrieret al.,2001).

This study showed that there was a highly significant elevation of Serum Iron and Serum Ferritin in the patient group compared to the control group. This was accompanied by subsequent reduction of TIBC in the patient group.

Oxidation of α-globin subunits leads to the formation of hemichromes, it bind to or modify various components of the mature red-cell membrane, such as protein band 3, protein 4.1, ankyrin, and spectrin. After precipitation of hemichromes, heme disintegrates, and toxic non- transferrin-bound iron species are released. The resulting free iron catalyzes the formation of reactive oxygen species (Rachmilewitz&Schrieret al., 2001).

Iron-dependent oxidation of membrane proteins and formation of redcell "senescence" antigens such as phosphatidylserine (Kuypers & Jong. 2004) cause thalassemic red cells to be rigid and deformed and to aggregate, resulting in premature cell removal (Tavazziet al.,2001).

Ineffective erythropoiesis and hepatosplenomegaly together result in Hypochromic microcytic anemia which in turn increases iron absorption plus transfusional iron overload both lead to increased levels of iron, ferritin and decreased TIBC (Porter, 2001).

The most common secondary complications are those related to transfusional iron overload, which can be prevented by adequate iron chelation. Iron-loading anemias are disorders in which hepcidin is regulated by opposing influences of ineffective erythropoiesis and concomitant iron overload (Pigeonet al.,2001).

The results of the present study revealed reduction of Hepcidin level in patient group compared to the control group this reduction was statistically significant. On the other hand, the ratio between hepcidin concentration and Serum Ferritin was highly reduced in patient group compared to the control group. This reduction was statistically significant.

The results showed that there was a positive correlation between hepcidin and Hb, PCV and MCV (i.e., as the value of hepcidin increases, the values of the Hb, PCV and MCV increase and vice versa) while there was a negative (i.e., inverse) correlation between hepcidin and (ferritin &Serum Iron) (i.e., as the values of hepcidin increase, the values of ferritin and Serum Iron decrease and vice versa).

Patients of β thalassemia major have decreased concentrations of hepcidin due to opposing influences of ineffective erythropoiesis and concomitant iron overload. This agreed with Wrighting and Andrews 2008 who reported that the erythroid regulator includes a mechanism to decrease hepcidin production. Accordingly, low hepcidin levels have been reported in patients with thalassemia and other disorders with ineffective erythropoiesis.

Wrighting and Andrews 2008 reported also that hepcidin expression was downregulated in a hepatocytic cell line after treatment with thalassemic sera.

This result was in agreement with Rund and Rachmilewitz 2005 who reported that Hepcidin levels were found to be low in patients with thalassemia intermedia and thalassemia major. Furthermore, serum from patients with thalassemia inhibited hepcidin messenger RNA expression in the HepG2 cell line, which suggests the presence of a humoral factor that downregulates hepcidin.

Zimmermann et al 2008 reported that hepcidin concentrations should be high in iron-loaded persons with β -thalassemia; however, hepcidin concentrations are low in these persons, unless they have recently received a transfusion. Production of growth differentiation factor 15 (GDF-15) by the expanded erythroid compartment contributes to iron overload in thalassemia by inhibiting hepcidin gene expression.

Nemeth and Ganz 2006 reported that when the ratio of urinary hepcidin to serum ferritin was analyzed as an index of appropriateness of hepcidin response to iron load, this ratio was still greatly decreased in thalassemia major patients when compared to normal subjects, indicating the continued regulation of hepcidin by a suppressive factor.

Pak et al 2006 reported that patients with β -thalassemia would be expected to have high hepcidin levels. To the contrary, patients with β- thalassemia have almost uniformly low urinary hepcidin. These and other clinical observations in iron-loading anemias would argue that erythropoiesis is able to suppress hepcidin production even in the face of severe iron overload.

Papanikolaou et al 2005 reported that hepcidin was measured in 8 patients with thalassemia major and 7 with thalassemia intermedia. Patients with thalassemia had very low urinary hepcidin levels, despite high serum ferritin levels that reflected systemic iron overload. Several patients with thalassemia had no detectable hepcidin.

The current study didn't agree with Origa et al 2007 who reported that hepcidin levels were elevated in thalassemia major, due to transfusions that reduce erythropoietic drive and deliver a large iron load, resulting in relatively higher hepcidin levels. In the presence of higher hepcidin levels, dietary iron absorption is moderated and macrophages retain iron, contributing to higher serum ferritin.

This result agreed with Brissot et al 2008 who reported that hepcidin deficiency in thalassemia major due to ineffective erythropoiesis leads to growth differentiation factor15 (GDF15) overexpression by the erythroblasts, which inhibits hepcidin expression. This could explain why (hepatocytic) iron overload can develop in thalassaemia in the absence of transfusions, and why hepcidin expression is relatively low in this disease despite transfusional iron excess (which should, by itself, lead to marked increased hepcidin expression).

Kemna et al 2008 reported that erythropoietin that stimulates erythropoietic activity has been shown to down-regulate liver hepcidin expression. However, in the absence of erythropoietic activity, hepcidin expression is no longer suppressed. The strong inverse association between erythropoietic drive and hepcidin production was also observed in several patients with congenital chronic anemias, which are characterized by low urinary hepcidin levels. The dotblot method was used to observe low/normal hepcidin levels for the degree of iron load in thalassemic patients.

Kattamis et al 2006 reported that urinary hepcidin was found to be suppressed in patients with thalassemia major. Tissue hypoxia triggers the production of EPO, which results in pronounced erythroid proliferation accompanied by increased sTfR levels. Hypoxia and yet-undefined signals from the robust erythroid activity down-regulate hepcidin production.

Nemeth and Ganz 2006 reported that Patients with chronic anemias with hemolysis or dyserythropoiesis, such as thalassemia syndromes suffer from iron overload. Measurements of urinary hepcidin in these patients indicated that hepcidin levels were severely decreased, despite systemic iron overload reflected by the patients’ elevated serum ferritin levels. Even in regularly transfused thalassemia patients, hepcidin levels were inappropriately low given the patients’ iron load, as indicated by the decreased ratio of urinary hepcidin to serum ferritin, used as an index of appropriateness of hepcidin response to iron load.

Toledano et al 2008 reported that Several diseases with chronic iron overload such as hereditary hemochromatosis and β-thalassemia major are characterized by low hepcidin expression in the liver. The low hepatic hepcidin in these patients is probably responsible for the intestinal absorption of iron.

Tanno et al 2007 also reported that Serum from thalassemia patients suppressed hepcidin mRNA expression in primary human hepatocytes, and depletion of GDF15 reversed hepcidin suppression. These results suggest that GDF15 overexpression arising from an expanded erythroid compartment contributes to iron overload in thalassemia syndromes by inhibiting hepcidin expression.

CONCLUSIONS

- Transfusional iron overload is the most common secondary complications in β thalassemia major.
- Hepcidin is a central regulator of iron homeostasis.
- Hepcidin concentration was decreased in the thalassemic group although elevated iron levels.
- Patients of β thalassemia major have decreased concentrations of hepcidin due to opposing influences of ineffective erythropoiesis and concomitant iron overload.

RECOMMENDATION

- Further study is recommended on large scale including the development of hepcidin- based diagnostics and therapies for iron-loading anemias that may offer more effective approaches to prevent the toxicity associated with iron overload.
- Further study is recommended to screen patients with β thalassemia who develop secondary iron overload to detect hepcidin level as in the future, therapeutic use of hepcidin and hepcidin agonists may help to restore normal iron homeostasis.
- Further study is recommended for evaluating and measuring hepcidin regulators as growth differentiation factor15 (GDF15) , Hemojuvelin (HJV) and others in cases of ineffective erythropoiesis and their effects on hepcidin expression.

SUMMARY

The thalassemias are a heterogeneous group of genetic disorders of haemoglobin synthesis , all of which result from a reduced rate of production of one or more of the globin chains of haemoglobin. The thalassemias are among the most common genetic disorders worldwide, occurring more frequently in the Mediterranean region, the Indian subcontinent, Southeast Asia, and West Africa.

The most common secondary complications are those related to transfusional iron overload, which can be prevented by adequate iron chelation.

The survival of individuals who have been well transfused and treated with appropriate chelation extends beyond age 30 years. Iron-chelation therapy is largely responsible for doubling the life expectancy of patients with thalassemia major.

Systemic iron is distributed among erythrocyte precursors in the bone marrow, tissue macrophages, liver, and all other tissues, with the largest amount found in circulating erythrocytes. Homeostasis is maintained by regulating the levels of plasma iron. Hepcidin, a circulating peptide hormone, has recently emerged as a key modulator of plasma iron concentration, and, thus, a central regulator of iron homeostasis.

Hepcidin binds to ferroportin and causes its internalization and degradation in lysosomes, thus effectively blocking the export of iron from the cells.

This study was conducted to measure hepcidin concentration in patients of β thalassemic major to explain its rule in iron metabolism for these patients who have iron overload.

This study was carried out in pediatric and clinical pathology department of Zagazig University Hospitals and included 30 β thalassemic major children and 10 apparently healthy children as a control group.

All the studied groups were subjected to:

- Full history taking and thorough clinical examinations .
- Laboratory investigations: C.B.C, Serum iron, Serum ferritin, TIBC, Hemoglobin electrophoresis, Liver function tests, kidney function tests and Hepcidin hormone was measured by Enzyme Linked Immunosorbant Assay in the serum.

From the results, it was found that:

- There was a highly significant difference between the two groups for Total Bilirubin and Direct Bilirubin .
- There was high significant difference between the two groups as regard SGPT, SGOT and Alkaline phosphatase.
- There was a highly significant difference between the two groups as regards RBCs, HCT, HB, MCV, MCHC and PDW. Also there was a significant difference for WBC and MCH.
- There was a highly significant difference between the two groups for Serum Iron, Serum Ferritin and TIBC.
- There was a highly significant difference between the two groups for HBA, HBA2 and HBF
- There was a highly significant increase in the control group compared to patient group for Hepcidin . Hepcidin concentration was decreased in the patients group although elevated iron levels compared to the control group who have normal iron levels and increased hepcidin concentration .
- The ratio between hepcidin and Serum Ferritin in the patients group was lower than that in the control group.
- There was a positive correlation between hepcidin in one side and Hb, Hct, and Mcv in the other side. Also there was a negative correlation between hepcidin and serum ferritin.

REFERENCES

A: Abboud S and Haile D J (2000). A novel mammalian iron-regulated protein involved in intracellular iron metabolism. J. Biol. Chem., 275, pp. 19906-19912.

Adamsky K, Weizer O, Amariglio N et al. (2004). Decreased hepcidin mRNA expression in thalassemic mice. Br. J. Haematol.,124, pp. 123- 124.

Aessopos A, Stamatelos G, Skoumas V et al. (1995). Pulmonary hypertension and right heart failure in patients with ß-thalassemia intermedia. Chest, 107, pp. 50-53.

Ahmad K A, Ahmann J R, Migas M C et al. (2002). Decreased liver hepcidin expression in the knockout mouse. Blood Cells Mol. Dis., 29, pp. 361-6.

Anderson G J, Darshan D, Wilkins S J et al. (2007). Regulation of systemic iron homeostasis: how the body responds to changes in iron demand. Biometals,20, pp.665-674.

Andrews N C (1999): Disorders of iron metabolism. N Engl J Med., 341(26), pp. 1986-1995.

Andrews N C (2000). Iron metabolism: Iron deficiency and iron overload. Annu. Rev. Genomics Hum. Genet.,1, pp. 75-98.

Angelucci E, Bai H, Centis F, et al (2002). Enhanced macrophagic attack on -thalassemia major erythroid precursors. Haematologica, 87, pp. 578-583.

B: Babitt, J L, Huang, F W, Wrighting, D M et al. (2006). Bone morphogenetic protein signaling by hemojuvelin regulates hepcidin expression. Nature Genetics, 38, pp. 531-539.

Badens C, Mattei M G, Imbert A M et al. (2002). A novel mechanism for thalassaemia intermedia. Lancet,359, pp. 132-133.

Barisani1 D, Pelucchi S, Mariani R, et al. (2008). Hepcidin and iron- related gene expression in subjects with Dysmetabolic Hepatic Iron Overload. Journal of Hepatology, 49(1), pp. 123-133.

Beutler E, Gelbart T, Lee P et al. (2000). Molecular characterization of a case of a transferrinemia. Blood, 96, pp. 4071-4074.

Borgna-Pignatti C, Rugolotto S, De Stefano P et al. (2004): Survival and complications in patients with thalassemia major treated with transfusion and deferoxamine. Haematologica,89, pp. 1187-1193.

Borregaard N, and Cowland J B (2006). Neutrophil gelatinase -associated lipocalin, a siderophore-binding eukaryotic protein. Biometals,19, pp. 211-215.

Bouva M J, Harteveld C L, van Delft, et al. (2006). Known and new delta globin gene mutations and their diagnostic significance. Haematologica, 91(1), pp. 129-32.

Bradai M, Abad M T, Pissard S, et al. (2003). Hydroxyurea can eliminate transfusion requirements in children with severe beta-thalassemia. Blood,102, pp. 1529-30.

Breda L, Gardenghi S, Guy E, et al. (2005). Exploring the role of hepcidin, an antimicrobial and iron regulatory peptide, in increased iron absorption in beta-thalassemia. Ann. NY Acad. Sci., 1054, pp. 417-422. Bridle KR, Frazer DM, Wilkins SJ, et al. (2003). Disrupted hepcidin regulation in HFE-associated haemochromatosis and the liver as a regulator of body iron homoeostasis. Lancet,361, pp. 669-673. Brissot P, Troadec MB, Bardou-Jacquet E, et al. (2008). Current approach to hemochromatosis. Blood Reviews, 22(4), pp. 195-210.

Britton RS, Fleming RE, Parkkila S, et al. (2002). Pathogenesis of hereditary hemochromatosis: Genetics and beyond. Semin Gastrointest Dis, 13, pp. 68-79.

C: Canonne-Hergaux F, Donovan A, Delaby C, et al. (2006). Comparative studies of duodenal and macrophage ferroportin proteins. Am. J. Physiol. Gastrointest. Liver Physiol., 290, pp. G156-G163.

Canonne-Hergaux F, Zhang A S, Ponka P, et al. (2001). Characterization of the iron transporter DMT1 (NRAMP2/DCT1) in red blood cells of normal and anemic mk/mk mice. Blood, 98, pp. 3823-3830. Cappellini M

D, Cohen A, Piga A, et al. (2006). A phase 3 study of deferasirox (ICL670), a once-daily oral iron chelator, in patients with beta-thalassemia. Blood, 107(9), pp. 3452-3455.

Ceci A, Baiardi P, Felisi M et al. (2002). The safety and effectiveness of deferiprone in a large-scale, 3-year study in Italian patients. Br J Haematol, 118, pp. 330-6.

Centis F, Tabellini L, Lucarelli G, et al (2000). The importance of erythroid expansion in determining the extent of apoptosis in erythroid precursors in patients with -thalassemia major. Blood, 96, pp. 3624- 3629.

Chan L N L, and Gerhardt E M (1992). Transferrin receptor gene is hyperexpressed and transcriptionally regulated in differentiating erythroid cells. J. Biol. Chem., 267, pp. 8254-8259.

Codrington J F, Li H W, Kutlar F, et al. (1990). Observations on the levels of Hb A2 in patients with different β -thalassemia mutations and a d chain variant. Blood, 76, pp. 1246- 1249.

Cohen A R, Galanello R, Pennell D J. et al., (2004). Thalassemia. Hematology. Am Soc Hematol Educ Program, 1, pp. 14-34.

Craig J E, Kelly S J, Barnetson R, et al. (1992). Molecular characterization of a novel 10.3 kb deletion causing β -thalassaemia with unusually high Hb A2. Br J Haematol, 82, pp. 735- 744.

Cunningham M J, Macklin E A, Neufeld E J, et al. (2004). Thalassemia Clinical Research Network. Complications of beta-thalassemia major in North America. Blood, 104(1), pp. 34-39.

D: Dean A, Couloumbier D and Brendel K(1994).Epi-info version,6.02 award processing data base and statistic program for Epidemiology on microcomputer,CDC,Atlanta,Georgia,USA.

De Domenico I, Ward D M, di Patti M C, et al. (2007). Ferroxidase activity is required for the stability of cell surface ferroportin in cells expressing GPI-ceruloplasmin. EMBO J., 26, pp. 2823-2831. De Maria R, Testa U, Luchetti L, et al (1999). Apoptotic role of Fas/Fas ligand system in the regulation of erythropoiesis. Blood, 93, pp. 796- 803.

Delaby C, Pilard N, Goncalves A S, et al. (2005). Presence of the iron exporter ferroportin at the plasma membrane of macrophages is enhanced by iron loading and down-regulated by hepcidin. Blood, 106, pp. 3979-3984.

Detivaud L, Nemeth E, Boudjema K, et al. (2005). Hepcidin levels in humans are correlated with hepatic iron stores, hemoglobin levels, and hepatic function. Blood,106, pp. 746-8.

Deugnier Y, Brissot P, and Loreal O (2008). Iron and the liver: Update 2008: A review. Journal of Hepatology, 48, pp. S113-S123. Divoky V, Indrak K, Mrug M et al. (1996). A novel mechanism of β thalassemia: the insertion of L1 retrotransposable element into β globin IVS II. Blood, 88, pp. 148.

Donovan A, Lima C A, Pinkus J L, et al. (2005). The iron exporter ferroportin/Slc40a1 is essential for iron homeostasis. Cell Metab., 1, pp. 191-200.

Du Z D, Roguin N, Milgram E, et al. (1997). Pulmonary hypertension in patients with thalassemia major. Am Heart J., 134, pp. 532- 537. E: El Beshlawy A, Galila M, Esmatet A, et al (2008). Assessment of puberty in relation to L-carnitine and hormonal replacement therapy in beta-thalassemic patients. Journal of Tropical Pediatrics, 54(6), pp. 375- 381.

El Beshlawy A, Kaddah, N, Rageb L, et al. (1999). Thalassemia Prevalence and Status in Egypt. European Society for PediatricHematology and Immunology, 45(5), pp. 760.

Eldor A and Rachmilewitz E A (2002). The hypercoagulable state in thalassemia. Blood, 99(1), pp. 36-43.

F: Fei Y J, Stoming T A, Kutlar A, et al., (1989). One form of inclusion body β -thalassemia is due to a GAAÆTAA mutation at codon 121 of theßchain. Blood, 73, pp. 1075-1077.

Finch, C. (1994). Regulators of iron balance in humans. Blood, 84, pp. 1697-1702.

Finch CA and Huebers HA(1986): Iron metabolism. Clin.Physiology.Biochem.vol 4,PP.5-10.

Fleming R E and Bacon B R (2005). Orchestration of iron homeostasis. N Engl J Med, 352(17), pp. 1741-1744.

Forget B G (2001). Molecular Genetics of the Human Globin Genes. In: Steinberg MH, Forget BG, Higgs DR, Nagel RL, editors. Disorders of

Hemoglobin: Genetics, Pathophysiology, and Clinical Management, 1st Ed., Cambridge, UK: Cambridge University Press , pp. 117-30. Franchini M, Veneri D (2004). Iron-chelation therapy: an update. Hematol J., 5(4), pp. 287- 292.

Fucharoen S, Ketvichit P, Pootrakul P, et al. (2000). Clinical manifestation of beta-thalassemia/hemoglobin E disease. Journal ofPediatric Hematology/Oncology, 22(6), pp. 552-557.

G: Ganz, T. (2003). Hepcidin, a key regulator of iron metabolism and mediator of anemia of inflammation. Blood, 102(3), pp. 783-788. Gardenghi S, Marongiu M F, Ramos P, et al. (2007). Ineffective erythropoiesis in {beta}-thalassemia is characterized by increased iron absorption mediated by down-regulation of hepcidin and up-regulation of ferroportin. Blood, 109(11), pp. 5027-5035.

Gaziev J, and Lucarelli G (2003). Stem cell transplantation for hemoglobinopathies. Curr Opin Pediatr, 15, pp. 24-31.

Giordano PC, Harteveld CL, Haak HL, et al. (1998). A case of non- β - globin gene linked β thalassaemia in a Dutch family with two additional a-gene defects: the common-a3.7 deletion and the rare IVS1-116 (AÆG) acceptor splice site mutation. Br J Haematol, 103, pp. 370-376. Gonzalez-Redondo JM, Stoming TA, Kutlar A, et al. (1989). A CÆT substitution at nt -101 in a conserved DNA sequence of the promoter region of the β -globin gene is associated with 'silent' β -thalassemia. Blood, 73, pp. 1705- 1711.

H: Handin I, Lux E, Stoseel T (2003). Hemoglobin Synthesis; In Handin RI, Lux SE, Stossel TP, eds. Principles and Practice of Hematology, 2nd ed. Lippincott Williams & Wilkins, pp. 1503-1513.

Hardison R (2001). Organization, Evolution, and Regulation of the Globin Genes. In: Steinberg MH, Forget BG, Higgs DR, Nagel RL, eds.

Disorders of Hemoglobin: Genetics, Pathophysiology, and Clinical Management, 1st Ed. Cambridge, UK: Cambridge University Press; pp. 95-116.

Harris Z L, Durley A P, Man T K, et al. (1999). Targeted gene disruption reveals an essential role for ceruloplasmin in cellular iron efflux. Proc. Natl. Acad. Sci., USA, 96, pp. 10812-10817.

Helms RA, Herfinda ET, Quan DJ, et al., (2006). Other Anemias. In: Helms RA and Quan DJ, Textbook of Therapeutics: Drug and Disease Management, 8th ed. Lippincott Williams & Wilkins, pp. 1813-1815. Hershko C, Konijn A M, and Link G (1998). Iron chelators for thalassaemia. Br. J. Haematol., 101, pp. 399-406.

Hillman RS, Ault KA, and Rinder HM (2005). Thalassemia. In: Hillman RS, Ault KA and Rinder HM,Hematology in clinical practice. 4th ed. McGraw-Hill Professional, pp. 65-72.

Hoffbrand A V, (2006). Essential Haematology. Essential Series, Moss PA H and Pettit JE, eds. 5th ed. Blackwell Pub, Alabama, USA, pp. 72-92. Huebers HA, Eng MJ and Josephŏon BM (1987). Plasma Iron and Transferrin Iron Binding Capacity Evaluated by Colorimetric and Immuno Precipitation Methods. Clinical Chemistry,33, pp.273-277. I: Iolascon A, d’Apolito M, Servedio V, et al. (2006). Microcytic anemia and hepatic iron overload in a child with compound heterozygous mutations in DMT1 (SCL11A2). Blood, 107, pp. 349-354. J: Jabado N, Canonne-Hergaux F, Gruenheid S, et al. (2002). Iron transporter Nramp2/DMT-1 is associated with the membrane of phagosomes in macrophages and Sertoli cells. Blood, 100, pp. 2617- 2622.

Jenkins Z A, Hagar W, Bowlus C L, et al. (2007). Iron homeostasis during transfusional iron overload in betathalassemia and sickle cell disease: Changes in iron regulatory protein, hepcidin, and ferritin expression. Pediatr. Hematol. Oncol., 24, pp. 237-243.

K: Kattamis A, Papassotiriou I, Palaiologou D, et al. (2006). The effects of erythropoetic activity and iron burden on hepcidin expression in patients with thalassemia major. Haematologica, 91(6), pp. 809-812. Kemna E H J M, Tjalsma H, Willems H L, et al. (2008). Hepcidin from discovery to differential diagnosis. Haematologica, 93 (1), pp. 90-97. Knutson M, and Wessling-Resnick M (2003). Iron metabolism in the reticuloendothelial system. Crit. Rev. Biochem. Mol. Biol., 38, pp. 61- 88.

Kozyraki R, Fyfe J, Verroust P J, et al. (2001). Megalin-dependent cubilin-mediated endocytosis is a major pathway for the apical uptake of transferrin in polarized epithelia. Proc. Natl. Acad. Sci., USA, 98, pp. 12491-12496.

Kristiansen M, Graversen J H, Jacobsen C, et al. (2001). Identification of the haemoglobin scavenger receptor. Nature, 409, pp. 198-201. Kuypers FA, de Jong K (2004). The role of phosphatidylserine in recognition and removal of erythrocytes. Cell Mol Biol, 50, pp. 147-158. L: La Nasa G, Argiolu F, Giardini C, et al. (2005). Unrelated bone marrow transplantation for beta-thalassemia patients: The experience of the Italian Bone Marrow Transplant Group. Ann N Y Acad Sci., 1054, pp. 186- 195.

Locatelli F, Rocha V, Reed W, et al. (2003). Related umbilical cord blood transplantation in patients with thalassemia and sickle cell disease. Blood, 101, pp. 2137-2143.

Loreal O, Haziza-Pigeon C, Troadec M B, et al. (2005). Hepcidin in iron metabolism. Curr Protein Pept Sci, 6, pp. 279-291.

M: Maquat LE (1995). When cells stop making sense: effects of nonsense codons on RNA metabolism in vertebrate cells. RNA, 1, pp. 453-65. Marini MG, Asunis I, Porcu L, et al (2004). The distal β -globin CACCC box is required for maximal stimulation of the β -globin gene by EKLF. Br J Haematol, 127, pp. 114-117.

Mathias LA, Fisher TC, Zeng L, et al (2000). Ineffective erythropoiesis in -thalassemia major is due to apoptosis at the polychromatophilic normoblast stage. Exp Hematol, 28, pp. 1343-1353.

McKie A T, Barrow D, Latunde-Dada G O, et al. (2001). An iron- regulated ferric reductase associated with the absorption of dietary iron. Science, 291, pp. 1755-1759.

Mims M P, Guan Y, Pospisilova D, et al. (2004). Identification of a human mutation of DMT1 in a patient with microcytic anemia and iron overload. Blood, 105(3), pp. 1337-1342.

Moreno PR, Purushothaman KR, Fuster V, et al. (2004). Plaque neo- vascularization is increased in ruptured atherosclerotic lesions of human aorta: implications for plaque vulnerability. Circulation, 110(14), pp. 2032-2038.

Muckenthaler M, Roy C N, Custodio A O, et al. (2003). Regulatory defects in liver and intestine implicate abnormal hepcidin and Cybrd1 expression in mouse hemochromatosis. Nat. Genet., 34, pp. 102-107.

Muñoz M, Villar I, García-Erce JA (2009). An update on iron physiology. World J Gastroenterol, 15(37), pp 4617-4626.

N: Nancy F O and Livieri M D (1999). The β-thalassemias. N Engl J Med., 341(2), pp. 99-109.

Nemeth, E and Ganz, T (2006). Regulation of iron metabolism by hepcidin. Annual review of Nutrition, 26, pp. 323-342.

Nemeth E, Tuttle M S, Powelson J, et al. (2004). Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science, 306, pp. 2090-2093

Nemeth E, Valore E V, Territo M, et al. (2003). Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein. Blood, 101, pp. 2461-2463.

Nicolas G, Chauvet C, Viatte L, et al. (2002). The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation. J. Clin. Invest., 110, pp. 1037-1044.

Niederau C, Fischer R, Purschel A, et al. (1996). Long-term survival in patients with hereditary hemochromatosis. Gastroenterology, 110, pp. 1107- 1179.

O: Offbrand A V, Cohen A, Hershko C (2003). Role of deferiprone in chelation therapy for transfusional iron overload. Blood, 102, pp. 17-24. Old J M, Olivieri N F and Thein S L (2001). Diagnosis and management of thalassaemia. In: Weatherall DJ, Clegg B, eds. The thalassaemia syndromes. 4th ed. Oxford, England, pp. 45-55.

Olivieri N F, Brittenham G M, McLaren C E, et al. (1998). Long-term safety and effectiveness of iron-chelation therapy with deferiprone for thalassemia major. N Engl J Med., 339, pp. 417-422.

Origa, R, Galanello, R, Ganz, T et al. (2007). Liver iron concentrations and urinary hepcidin in ß-thalassemia. Haematologica, 92(5), pp. 583- 588.

Orofino M G, Argiolu F, Sanna M A et al., (2003). Fetal HLA typing in beta thalassaemia: implications for haemopoietic stem-cell transplantation. Lancet, 362, pp.41-42.

P: Pace BS and Zein S (2006). Understanding mechanisms of gamma- globin gene regulation to develop strategies for pharmacological fetal hemoglobin induction. Dev Dyn, 235, pp. 1727-1737.

Pak M, Lopez M A, Gabayan V, et al., (2006). Suppression of hepcidin during anemia requires erythropoietic activity. Blood, 108(12), pp. 3730-3735.

Papanikolaou G, Samuels M E, Ludwig E H, et al. (2004). Mutations in HFE2 cause iron overload in chromosome 1q linked juvenile hemochromatosis. Nat. Genet., 36, pp. 77-82.

Papanikolaou G, Tzilianos M, Christakis J I, et al. (2005). Hepcidin in Iron Overload Disorder. Blood, 105, pp. 4103-4105

Park C H, Valore E V, Waring, et al. (2001). Hepcidin, a urinary antimicrobial peptide synthesized in the liver. J Biol Chem, 276, pp. 7806-7810.

Parkes JG, Hussain RA, Olivieri N F, et al. (1993). Effects of iron loading on uptake, speciation, and chelation of iron in cultured myocardial cells. J Lab Clin Med, 122, pp. 36-47

Persons D A, Nienhuis A W (2003). Gene therapy for the hemoglobin disorders. Curr Hematol Rep, 2: 348-355.

Pietrangelo A. (2007). Haemochromatosis: an endocrine liver disease. Hepatology, 46, pp. 1291-1301.

Pigeon C, Ilyin G, Courselaud B, et al. (2001). A new mouse liver- specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload. J. Biol. Chem., 276, pp. 7811-7819.

Porter JB (2001). Practical management of iron overload. Br J Haematol; 115(2) , pp. 239-252.

Puthenveetil G, Malik P (2004). Gene therapy for hemoglobinopathies: are we there yet?. Curr Hematol Rep, 3, pp. 298-305.

Q: Qiu A, Jansen M, Sakaris A, et al. (2007). Identification of an intestinal folate transporter and the molecular basis for hereditary folate malabsorption. Cell, 127(5), pp. 917-928.

R: Rachmilewitz EA, Schrier S (2001). The pathophysiology of - thalassemia. In: Steinberg MH, Forget BG, Higgs DR, Nagel RL, eds. Disorders of hemoglobin: genetics, pathophysiology, and clinical management. Cambridge, England: Cambridge University Press, pp. 233-251.

Rimoin DL, Emery AE, Michael Connor J et al, (2006). Hemoglobinopathesis and Thalassemia. In: Emery and Rimoin'sPrinciples and Practice of Medical Genetics, 5th ed. Elsevier Health Sciences, pp.1665.

Rivera S, Liu L, Nemeth E, et al. (2005a). Hepcidin excess induces the sequestration of iron and exacerbates tumor-associated anemia. Blood, 105, pp. 1797-1802.

Rivera S, Nemeth E, Gabayan V, et al. (2005b). Synthetic hepcidin causes rapid dose-dependent hypoferremia and is concentrated in ferroportin-containing organs. Blood, 106, pp. 2196-2199.

Rund D and Rachmilewitz E (2005). Beta-thalassemia. N Engl J Med., 353(11), pp. 1135-1146.

S: Sabella C and Cunningham RJ (2006). Pediatric Hematology Oncology. In: Sabella C and Cunningham RJ,The Cleveland Clinicintensive review of pediatrics., 2nd edition. Lippincott Williams & Wilkins. PP.468-472

Sacher A, Cohen A, and Nelson N (2001). Properties of the mammalian and yeast metalion transporters DCT1 and Smf1p expressed in Xenopus laevis oocytes. J. Exp. Biol., 204, pp. 1053-1061.

Sampietro M, Lupica L, Perrero L, et al (1997). The expression of uridine diphosphate glucuronosyltransferase gene is a major determinant of bilirubin level in heterozygous β -thalassaemia and in glucose-6- phosphate. Br J Haematol, 99, pp. 437-439.

Sheftel AD, Zhang AS, Brown C, et al. (2007). Direct interorganellar transfer of iron from endosome to mitochondrion. Blood, 110, pp. 125 - 132.

Sorrentino BP, Niehuis AW (2001). Prospects for gene therapy of sickle cell disease and thalassemia. In:Disorders of Hemoglobin. By Steinberg MH, Forget BG, Higgs DR, Nagel RL (eds): Genetics, Pathophysiology, and Clinical Management. Cambridge University Press, (2nd ed). pp. 1084-118.

T: Tai DYH, Wang YT, Lou J, et al. (1996). Lungs in thalassaemia major patients receiving regular transfusion. Eur Respir J., 9, pp. 1389-1394. Taketani S (2005). Aquisition, mobilization and utilization of cellular iron and heme: endless findings and growing evidence of tight regulation. Tohoku J Exp Med, 205(4), pp. 297-318.

Talwar GP and Srivastava LM (2004). The Haematopiotic System. In: Talwar GP, Srivastava LM, Textbook of Biochemistry and Human Biology. 3rd edition, PHI Learning Pvt. Ltd, PP.213-214.

Tamary H, Shalev H, Perez-Avraham G, et al. (2008) Elevated growth differentiation factor 15 expression in patients with congenital dyserythropoietic anemia type I. Blood,112, PP. 5241-5244. Tanner MA, Galanello R, Dessi C. et al., (2007). A randomized, placebo- controlled, double-blind trial of the effect of combined therapy with deferoxamine and deferiprone on myocardial iron in thalassemia major using cardiovascular magnetic resonance. Circulation. 115, pp. 1876- 1884.

Tanno, T, Bhanu, N V, Oneal, P A et al. (2007). High levels of GDF15 in thalassemia suppress expression of the iron regulatory protein hepcidin. Nature Medicine, 13, pp. 1096-1101.

Tavazzi D, Duca L, Graziadei G, et al. (2001). Membrane-bound iron contributes to oxidative damage of -thalassemia intermedia erythrocytes. Br J Haematol, 112, pp. 48-50.

Testa U (2004). Apoptotic mechanisms in the control of erythropoiesis. Leukemia, 18, pp. 1176-1199.

Thein SL (1998). β Thalassaemia. In: Rodgers GP, ed. Baillière's Clinical Haematology, Sickle Cell Disease and Thalassaemia. London: Baillière Tindall, pp. 91-126.

Thein SL (2001). Structural Variants with a β thalassemia phenotype. In: Steinberg MH, Forget BG, Higgs DR, Nagel RL, editors. Disorders of Hemoglobin: Genetics, Pathophysiology, and Clinical Management. Cambridge,UK: Cambridge University Press, Cambridge, UK, pp. 342- 55.

Toledano, M., Kozer, E., Goldstein, L H et al. (2009). Hepcidin in acute iron toxicity. The American Journal of Emergency Medicine, 27(7), pp. 761-764.

Trenor CC, Campagna DR, Sellers V M. et al (2000). The molecular defect in hypotransferrinemic mice. Blood, 96, pp. 1113-1118. Trifillis P, Ioannou P, Schwartz E, et al (1991). Identification of four novel δ β -globin gene mutations in Greek Cypriots using polymerase chain reaction and automated fluorescence-based DNA sequence analysis. Blood, pp. 298-305.

V: Valeri CR, Bond JC and Flower K. (1965). Quantitation of Serum Hemoglobin-Binding Capacity using Cellulose Acetate Membrane Electrophoresis. Clinical Chemistry, 11, pp.581-588.

Viatte L, Lesbordes-Brion JC, Lou DQ, et al. (2005). Deregulation of proteins involved in iron metabolism in hepcidin-deficient mice. Blood, 105(12), pp. 4861-4864.

Vulpe CD, Kuo YM, Murphy TL, et al. (1999). Hephaestin, a ceruloplasmin homologue implicated in intestinal iron transport, is defective in the sla mouse. Nat. Genet., 21, pp. 195-199.

W: Walters MC, Quirolo L, Trachtenberg ET et al., (2005). Sibling donor cord blood transplantation for thalassemia major: Experience of the Sibling Donor Cord Blood Program. Ann N Y Acad Sci., 1054, pp. 206-213.

Wanless IR, Sweeney G, Dhillon AP, et al. (2003). Lack of progressive hepatic fibrosis during long-term therapy with deferiprone in subjects with transfusion-dependent b-thalassemia. Blood, 100, pp. 1566-1569.

Weatherall D J and Clegg J B (2001). Inherited haemoglobin disorders: an increasing global health problem. Bull. World Health Organ., 79, pp. 704-712.

Weatherall DJ and Clegg JB (2000). The Thalassemia Syndromes. 4th ed., Oxford, UK, Blackwell.

Weinstein D A, Roy C N, Fleming M D, et al. (2002). Inappropriate expression of hepcidin is associated with iron refractory anemia: Implications for the anemia of chronic disease. Blood, 100, pp. 3776- 3781.

Weizer-Stern O, Adamsky K, Amariglio N (2006). Downregulation of hepcidin and haemojuvelin expression in the hepatocyte cell-line HepG2 induced by thalassaemic sera. Br J Haematol., 135(1), pp. 129-138.

Wonke B (2001). Clinical management of b-thalassemia major. Semin Hematol., 38, pp. 350-359.

Wood W G (2001). Hereditary Persistence of Fetal Hemoglobin and δ β Thalassemia. In: Steinberg MH, Forget BG, Higgs DR, Nagel RL, eds.

Disorders of Hemoglobin: Genetics, Pathophysiology, and Clinical Management. Cambridge, UK: Cambridge University Press, pp. 356- 388.

Wrighting D M and Andrews N C (2008). Iron Homeostasis and Erythropoiesis. Current Topics in Developmental Biology, 82, pp. 141- 167.

Wu K H, Chang J S, Tsai C H et al. (2004). Combined therapy with deferiprone and desferrioxamine successfully regresses severe heart failure in patients with beta-thalassemia major. Ann Hematol., 83, pp. 471-473.

X: Xu H, Jin J, DeFelice L J, et al (2004). A spontaneous, recurrent mutation in divalent metal transporter-1 exposes a calcium entry pathway. Biol., 2, , pp. 50.

Y: Yachie A, Niida Y, Wada T, et al. (1999). Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase-1 deficiency.

J. Clin. Invest., 103, pp. 129-135.

Yaish H M (2007). Thalassemia. Available at:Thalassemia Overview - eMedicine Pediatrics General Medicine, retrieved from www.emedicine .com.

Z: Zhang AS,Yang F, Meyer K, et al. (2008). "Neogenin-mediated hemojuvelin shedding occurs after hemojuvelin traffics to the plasma membrane". J. Biol. Chem. 25, pp. 17494-502.

Zimmermann, M B, Fucharoen, S, Winichagoon, P, et al. (2008). Iron metabolism in heterozygotes for hemoglobin E (HbE), -thalassemia 1, or β-thalassemia and in compound heterozygotes for HbE/β-thalassemia. American Journal of Clinical Nutrition, 88(4), pp. 1026-1031. Zoller H, and Cox T M (2005). Hemochromatosis: Genetic testing and clinical practice. Clin. Gastroenterol. Hepatol., 3, pp. 945-958.

illustration not visible in this excerpt